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Immunoperoxidase staining for cytokeratin using the microwave oven

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Quinnipiac
College
IMMUNOPEROXIDASE STAINING FOR CYTOKERATIN
USING THE MICROWAVE OVEN
By
Susan M. Mitchell
B .A . Assumption College,
1984
A THESIS
Presented to the School of Allied Health and Natural Sciences
and Quinnipiac College
in partial fufillment of the requirements
for the degree of
Master of Health Science
May 1986
S'
m
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ABSTRACT
IMMUNOPEROXIDASE STAINING FOR CY TOKERATIN
USING THE MICROWAVE OVEN
Susan M. Mitchell
Master of Health Science
School of A l l i e d Health and Natural Science
Q u i n nip iac College
May 19 8 6
Cytokeratin is a structural protein found primarily in
epithelial cells.
Its presence is used to identify the
origin of certain tumors.
Cytokeratin can be d ete c t e d by
the immunoperoxidase
technique,
(PAP)
a valuable but time
consuming procedure.
A microwav£ oven was utilized in this proje ct in an
attempt to decrease immunoperoxidase staining time.
Skin
of human thigh was stained with commercially pre pared PAP
reagents for cytokeratin incorporating brief m icrowave
exposures at certain steps.
The tolerance of P A P antibody
and enzyme reagents to microwave irradiation was evaluated.
Individual brief m icrowave treatments of sections
covered with PAP reagents was not harmful to the tissue or
reagents.
A 20 second microwave exposure, prior to routine
incubation of the primary antibody,
a n t i - c y t o k e r a t i n , was
found to significantly enhance the stain intensity. However,
Q U IN N IPIA C COLLEGE LIBRARY
HAMDEN, CT. 06518
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164 0
accumulations of repeated microwave treatments appeared
to destroy the tissue or the reagents, or both,
as reflected
by considerable non-specific background staining.
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IMMUNOPEROXIDASE STAINING FOR CYTOKERATIN
USING THE MICROWAVE OVEN
This thesis is approved as a credible and independent invest­
igation by a candidate for the degree of Master of Health Sciences,
and is acceptable as meeting the thesis requirements for this degree,
but without implying that the conclusions reached by the candidate
are necessarily the conclusions of the major department.
Qjinnipiac College Thesis Advisor
Irwin Beitch, Ph.D.
Professor of Biology
Department of Medical Laboratory Sciences
Quinnipiac College
Clinical Thesis Advisor
Leo J. Kelly, M.H.S.
Clinical Coordinator - Pathologists' Assistants Training
Program, Quinnipiac College and West Haven Veterans
Administration Medical Center
Medical Director
R jtvscb.
___________________
Pathologists' Assistants Training Program
Rosa E. Enriquez, M.D.
Director of Pathologists' Assistants Training Program
West Haven Veterans Administration Medical Center
Assistant Professor of Pathology, Yale University
Quinnipiac College
Edward Kavanagh, Ph.D.
Assistant Professor of Biology
ii
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ACKNOWLEDGEMENTS
I wish to express my appreciation to the following
individuals for their help and assistance in the completion
of this thesis project.
To Rosa Enriquez,
M . D . , whose guidance and professional
knowledge played a major role in the initiation and form­
ulation of this project.
To Leo Kelly,
patience,
M . H . S . , who generously offered his time,
and insight to me throughout this project,
as well
as over the past two years.
To Irwin Beitch,
P h . D . , for sharing his kn owledge and
experience to help d evelop a scientifically sound research
project and a well wr it t e n paper.
To Kent Marshall,
Ph.D.
and Edward K a v a n a g h , P h . D . , for
their technical guidance and knowledge that were essential
for proper completion of this project.
To my parents,
continuous
interest,
my endeavors,
Mr. and Mrs. Carl M i t c h e l l , for their
support,
and encouragement through all
especially over the past two years.
i ii
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TABLE OF CONTENTS
Page
I N TRO DUC TI ON................................................... 1
Statement of the P r o j e c t ................................1
Literature R e v i e w ........................................ 2
Microwave Energy and H e a t i n g ..................... 2
The Effect of Microwave Radiation
on Biological M o l e c u l e s ..................... 3
Laboratory Use of a Microwave O v e n .............. 6
Immunocytochemis t r y ................................7
C y t o k e r a t i n ........................................ 11
The S k i n ........................................... 13
Immunoperoxidase S t a i n i n g ........................15
Peroxidase-Antiperoxidase Staining
for C y t o k e r a t i n ............................. 17
R a ti on ale ................................................ 21
MATERIALS AND M E T H O D S ........................................ 24
R E S U L T S ........................................................ 31
Effects of Microwave Treatment on
Individual PAP Serum R e a g e n t s ......................... 31
Physical T o l era nce of Tissue Sections and
PAP Serum Reagents to Microwave E x p o s u r e ...... 31
Individual Micr owa ve Irradiation of PAP
Serum Reagents With Subsequent
Routine I n c u b a t i o n s ...............................31
Microwave Irradiation of PAP Serum Reagents
Without Routine I n c u b a tio ns ..................... 33
Effects of Microwave Treatment on Combinations
of PAP Serum Reagent S t e p s ............................ 33
iv
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TABLE OF CONTENTS
(continued)
Page
Combinations of Microwave Treatments Which
Were Shown to Give Positive Results
I n d i v i d u a l l y........................................33
Microwave Treatment of Ail PAP Serum
Reagents Except the Primary A n t i b o d y ............... 39
Microwave Staining of Five Surgical
Specimens Containing C yt oke rat in ................. 39
D I S C U S S I O N ......................................................46
Data A n a l y s i s ............................................ 46
Overall C o n c l u s i o n s ..................................... 56
RE F E R E N C E S ...................................................... 58
APPENDIX 1 ......................................................61
APPENDIX I I .....................................................62
v
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L IS T
OF T A B L E S
Page
Table 1.
A comparison of routine reaction times and
microwave reaction times for specific
steps in some staining p r o c e d u r e s ................. 8
Table 2.
Functions and properties of the
five classes of immunoglobulin p r o t e i n s ......... 12
Table 3.
Individual microwave irradiation of PAP
serum reagents with subsequent
routine i n c u b a t i o n s ................................ 3 2
Table 4.
Microwave irradiation of PAP serum
reagents
without subsequent routine in cub at ion s.......... 35
Table 5.
Further microwave testing of anti-cytokeratin
and peroxidase-antiperoxidase r e a g e n t s .......... 36
Table 6.
Methods used to reduce reaction
time of anti-cytokeratin r e a g e n t ................. 37
Table 7.
Combined microwave treat me nt PAP p r o c e d u r e .....3 8
Table 8.
Results obtained using combined microwave
treatment for all PAP serum r eag en ts ............ 40
Table 9.
Combined microwave tr eatment PAP procedure
for all steps except the primary
antibody s t e p ....................................... 41
9
Table 10. Results obtained using combined microwave
treatment for all PAP reagents
except a n t i - c y t o k e r a t i n ........................... 42
Table 11.
Comparison of routine and microwave
staining of specimens known to
contain c y t o k e r a t i n ................................ 45
vi
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LIST OF FIGURES
Page
s p e c t r u m ...................... 4
Figure 1.
The electromagnetic
Figure 2.
Schematic drawing of an antibody m o l e c u l e
Figure 3.
Cell layers of s k i n ............................... 16
Figure 4.
Schematic diagram of a peroxiaaseantiperoxidase m o l e c u l e .......................... 20
Figure 5.
Schematic diagram of the peroxidaseantiperoxidase staining p r oce du re .............. 22
vi i
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10
L IS T
OF
PLATES
Page
Plate 1.
Human skin from thigh stained by routine
PAP procedure for c y t o k e r a t i n ....................25
Plate 2.
Illustration of grading scale used to
evaluate specific c y t o k er at in stain
reaction in a PAP p r o c e d u r e ...................... 26
Plate 3.
Illustration of grading scale used to
evaluate non-specific background
staining in a PAP procedure
for cy tok er ati n .....................................27
Plate 4.
Comparison of routine and microwave PAP
procedure for cytokeratin staining
r e a c t i o n s ........................................... 34
Plate 5.
Comparison of a routine PAP procedure
reaction and a successful microwave
r e a c t i o n .............................................43
viii
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INTRODUCTION
Statement of the Project
The immunoperoxidase staining technique is a relatively
new procedure which uses antibodies to d emonstrate an­
tigenic substances in tissues.
It has become a highly
regarded tool to the pathologist.
veals about substances
in tissues can be a great aid in the
process of diagnosing disease.
ever,
The information it re­
One of its drawbacks, how­
is that it requires many hours to complete the pro­
cedure.
Microwave heating has been successfully used to
expedite some staining procedures other than immunoperox­
idase staining
(1,2,3) .
A project was design ed to explore
the possibilities of utilizing microwave energy to sig­
nificantly decrease the amount of time necessary to perform
this procedure.
The literature does not indicate that this
method has ever been attempted.
Special stains r outinely used in the laboratory
microscopically reveal distinct structures,
substances in tissues.
organisms, or
It is on the basis of this type
of information that pathologists confirm many disease
diagnoses.
The time necessary to complete some of these
diagnostic staining procedures can delay treatment and be
detrimental to the well- be ing of the patient.
importantly,
though,
Most
the degree of specificity of a stain
1
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determines its d ia g n o s t i c value.
is for a particular substance,
The more specific a stain
the more v alu ab le it is for
indicating the p res en ce of that substance.
The mechanism by w h i c h stains depict histopathology is
based on chemical rea c t i o n s between the stain and substances
in the tissue.
Because heat increases the rate of reaction
for some chemical reactions,
it can be used to speed up many
staining procedures.
The microwave oven is a fairly inexpen­
sive instrument which
provides heat quickly by greatly in­
creasing molecular m o v e m e n t in substances.
Its use in
speeding up i m mun ope ro xid as e staining procedures,
therefore,
seems practical.
Literature Review
A review of the li terature will be c o mp ris ed of inform­
ation on several subj ect s which will give a basis of under­
standing for this project.
is used in the mi c r o w a v e
other
histological
will be described.
Microwave ra diation and how it
(MW) oven will be discussed.
Some
procedures which ut ilize the MW oven
Immunocytochemistry and how it relates
to the immunoperoxidase staining procedure will be reviewed
with respect to stain ing for human cytoke rat in in skin sections.
Microwave Energy and H e a t i n g
The source of e n e r g y
for a MW oven is electromagnetic
radiation char ac ter ize d by a particular w av e l e n g t h described
as a microwave.
El ec tromagnetic radiation is defined as
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3
"energy that is transmitted through space in the form of
waves"
(4).
The size of the wavelength determines the d i f ­
ferent types of radiation
(see Figure 1).
Microwaves are
characterized by wavelengths varying from lO^nm to 10®nm,
as compared to visible light waves which range from 400nm to
750nm
(4).
The Effect of Microwave Radiation
on Biological Molecules
Microwaves are p r odu ce d in a MW oven by a tube called
a magnetron.
This occurs through the interaction of strong
magnetic and electric fields
(1).
In general, mic rowaves
are capable of interaction with dipolar molecules
ways:
in two
1) by imparting kinetic energy and raising temperature,
and 2) by altering electric fields
(5).
These o ccu r because
MW radiation causes d ipolar molecules to rapidly osc il lat e
180 degrees.
This causes
increased intramolecular and inter-
molecular motion, and as a result thermal energy
increases(5).
This energy is not enough to alter covalent bonds,
could readily interact w i t h steric bonds
bonds and Van der Waals
interactions)
for biological function
(5) .
but
(ie., h yd r o g e n
which are es sential
Dipolar water molecules and polar side chains of larger
proteins are the molecules in a cell which are a ffe ct ed
most by MW exposure
(6).
The basic principle on w h i ch
this MW interaction is ba sed is:
"whatever the mecha ni sms
of absorption of e le ctromagnetic radiation,
absorbed
must be thermalized"
(6).
the ene rg y
This means that the
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4
cosmic
and
gamma
waves
X-rays
ultra­
violet
waves
infrared
waves
micro­
waves
radiowaves
10
wavelength
Fig.
1.
(nm)
The electromagnetic spectrum.
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energy introduced to the cell system m u s t be accounted for,
once present.
It is the electrical c h a r a c t e r of the mol e­
cules which determines how they are a f f e c t e d by micro­
waves
(7) .
Many studies involving the exact ramifications of
microwave exposure to biological m o le cul es and cells have
been done.
Presently,
several theories exist,
involve a lte ration of cell function
(6,7) .
all of which
One theory is
that MW exposure causes a change in p o lar iz ati on at the
boundary of m aterials with different d i e l e c t r i c constants.
In a cell the mo st likely boundary is the cell membrane.
change in pol arization here alters the
lipia-water trans­
ition in the cell,
function
thus affecting cell
Another theory has a more structural basis.
A
(6).
It is
known that the macromolecules which make up the functioning
units of the cell
(eg., mitochondria and ribosomes)
depend
on correct p ositioning of neighboring m ol e c u l e s for sequential
reactions,
thus proper functioning
(6).
Some investigators
believe MW exposure causes the rotation of molecules in a
cell.
Therefore,
the altered position of molecules which
are adjacent to the macromolecules may interfere with the
normal functioning of a cell's organelles.
Experiments
particles,
involving the effect of microwaves on
in general,
position in cells
support the idea that molecular
is altered.
These studies have e s t a b ­
lished that m icrowave exposure causes
particles to form distinct patterns"
"randomly arranged
(7).
In other words,
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electromagnetic fields cause a rearrangement of particles
exposed to them.
Another mechanism by which it is felt that microwaves
alter cell function is based on the increased temperature
in a cell after MW exposure.
Taylor and Cheung proposed,
"Because of the approximately 100A thickness of the cell
membrane,
even a small absolute difference in temperature
be tween two sides of the membr ane results in a large thermal
gradi en t across the cell membrane"
then,
(6).
This gradient,
affects the flow of solutes and solvents across the
membrane.
In this way cellular function is altered.
Laboratory Use of a Microwave Oven
The microwave oven has been successfully used as a
heat source for other histological procedures including
fixation,
and metallic impregnation in some special stains
(1,2,3).
In reference to tissue fixation
(1,2),
the t i s ­
sues to be fixed are placed in saline or formalin,
then
heated in M W oven which is controlled to keep the t e m p ­
erature from rising above 62° C.
of tissue proteins,
and,
thus,
The result is co agu la tio n
fixation of the tissue for
histological purposes.
Another procedure successfully carried out in a MW
oven is metallic histological staining
metallic
Routinely,
impregnation for stains such as methena.T.ine silver
takes several hours to complete
heat,
(1,2,3).
(8).
Using MW produced
this staining is done in 60 to 75 seconds with
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results equal to or better than those of the routine method.
In particular, MW processed tissue has a reduced amount of
background staining
(1).
Some other specific procedures which have been docu­
mented as successful,
and a comparison of their routine and
MW reaction times are found in Table 1.
not commomly used in the laboratory,
for future use.
Although these are
they do hold possibility
As has been stated previously,
rapid pro­
cedures for a variety of special stains would gr ea tly aid
the pathologist in makin g diagnoses more quickly.
Immunocytochemistry
Antibodies are a diverse group of proteins w hich are
divided into five classes called immunoglobulins
(Ig).
though all immunoglobulins share many properties,
members of each class
function differently
(9) .
Al­
the
They are
all part of the humoral immune system of the body,
and aid
in the protection ag ai nst foreign substances.
Humoral immunity is that aspect of the immune system
which is mediated by cell products of lymphoid tissue
rather than the lymphocytes themselves
substance
(antigen)
(10).
Wh en a foreign
is introduced into the body,
it moves
throughout it and comes in contact with B lymphocytes in
lymph tissue.
Here the antigen programs u ncommitted B-cells
to produce specific antibody proteins against it.
These
B-cells then mature and become plasma cells, w h ich produce
the antibody molecules.
Whenever the original a ntigen is
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Table 1
A COMPARISON OF ROUTINE REACT IO N TIMES AND
MICROWAVE REACTION TI MES FOR
SPECIFIC STEPS IN SOME
STAINING PR OC EDURES
Name of stain and
step involved
Routine
reaction times
Microwave
reaction time
Dieterle silver impreg­
nation
40 min
50 sec
Enzymatic acid phospha­
tase incubation in
substrate
4 hrs
30 sec
Alcian blue staining
in alcian blue solution
30 min
5 0 sec
Perl's iron treatment in
ferrocyanide-hydrochloric acid solution
10 min
45 sec
Fontana-Masson silver
impregnation
34 hrs
75 sec
All routine reaction times are r ep or ted here according to
reference number 8.
All microwave reaction times are report ed here according to
reference number 2, except for the Dieterle stain, which is
from reference number 3.
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introduced to the body again,
the antibodies will be pr es ­
ent to begin the chain of events which will de str oy the
antigen
(10) .
The structure of all antibody molecules in general is
similar.
acids
They are composed of two light chains of amino
(molecular weight 22,500),
amino acids
and two heavy chains of
(molecular wei g h t 55,000)
(see Figure 2).
These four subunits are held together
and form Y- or T-shaped molecules
by disulf id e bonds,
(10).
The end of the
molecule with the light chains has terminal amino groups,
while the heavy chain end terminates with carboxyl groups.
Each of these ends has a specific role in immune reactions.
The two "arms" of the Y, each with a part of one
heavy chain and one light chain,
comprise two of the three
principle units of the antibody molecule.
the Fab units.
an antigen.
These are called
It is here where the antibody combines with
This is also the flexible part of the molecule
which allows for the shape variation;
a Y may become a T.
The third unit is the base of the molecule,
of the carboxyl ends of the two heavy chains.
the Fc unit.
It is called
Here antibodies can combine with other cells,
such as macrophages and complement proteins,
case with IgG.
composed
This aids
as is the
in the immune response to destroy
foreign substances in the body.
It is the varying sequences
of amino acids at both the Fab and the Fc ends w h ich d e t e r ­
mine the exact function of each
(9).
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] COOH
COOH
C O OH
COOH
Key:
= 1
light c hain
- heavy ch ain
nh
COOH
s
s
2 - amino end
- carboxyl end
- disulfide bond
F i g . 2. Schematic drawing of an antibody molecule, as
adapted from Immunology III, Bellanti, e d . (9)
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11
As has been mentioned,
molecules.
there are five classes of these
Each g r ou p differs in its general function; and,
with in each group the Fab portions of the molecules differ
to combine with d i f f er ent antigens.
referred to as IgG,
IgA,
IgM,
The five classes are
IgD, and IgE.
ular functions are listed in Table 2.
Their partic­
Type G immunoglob­
ulins are involved in the immunoperoxidase staining re­
actions .
Antibodies are formed against certain substances
found in tumors.
Then,
they can be used in the immunoper­
oxidase staining technique as tumor markers.
the nature, or origin,
In this way
of tumor cells can be identified.
This method of staining is very specific for the particular
antigens against w h i c h the immunoperoxidase antibodies are
made.
Cytokeratin
Cytokeratin is an antigenic substance found in tumors
w h ich are of epithelial origin.
It is a member of a family
of filamentous proteins called intermediate filaments, which
are characterized by a diameter of 8 to lOnm.
They function
as part of the cytosk el eto n of eukaryotic cells
(11,12).
These intermediate filaments are subdivided into five groups.
Each of these is found in a particular cell type as follows:
1) cytokeratin - epi thelial cells,
chymal cells,
2) vimentin - m e sen ­
3) de sm i n - muscle cells,
4) neurofilaments -
neurons, and 5) glial filaments - astrocytes
(11).
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Table 2
SOME FUNCTIONS AND PROPERTIES OF THE FIVE
CLASSES OF IMMUNOGLOBULIN PROTEINS
Class
Functions
/
Properties
IgG
fixes complement, crosses placenta, combats
mi croorganisms and toxins, combines with
Fab po rti on of another antibody / most
abundant, smallest
IgA
provides immunity in external secretory
systems of the body / most abundan t Ig in
sero-mucous secretions
IgM
fixes complement, reacts first in the line
of defense against bacteremia, acts intravascularly / largest
IgD
surface r ec ep tor on lymphocytes, probably
for ini ti ati on of immune response
IgE
combines w i t h mast cells and basophils /
responsible for signs of atopic allergy,
raised in parasit e infections
adapted from Bellanti,
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ed.
13
All these types c onsist
of a single polype pt ide unit,
except for cy t o k e r a t i n which consists of 19 polypeptides,
and are e xpressed differ ent ly by d if f e r e n t ep ithelial cell
types in the body
soluble,
(12) .
The cytokeratins are all water in­
i n t r a c e l l u a r , and are found in g re a t e s t abundance
in the cells of the epidermis and its adenexa.
Here they
are predominately responsible for c y t o a r c hi te ctu ral support.
Not all c yto ker at in subclasses will stain w i t h the anticytokeratin antibodies in a PAP for cy t o k e r a t i n procedure.
No other groups of intermediate filaments will stain pos­
itively with c y t o k e r a t i n pr o c e d u r e s .
The possible reas on that all 19 subclasses of cyto­
keratin do not stain with the same a nti - c y t o k e r a t i n anti­
bodies is related to different molecular w eig hts of the
peptides.
Each of the 19 cytokeratins has a characteristic
molecular we ig h t v ary ing from 37 to 68 kilodaltons.
example,
For
c y t o ke rat in formed by cells of a squamous cell tumor
has a much lower m o l ec ula r weight than the cyt ok era ti n which
forms over a callus on the sole of the foot.
An antibody
formed against one of these may not bind to the tissue of
the other due to the high degree of specifi cit y w h i c h is
characteristic of immune reactions
(13).
The Skin
The e p i th el ium of skin contains an a b un da nce of cyto­
keratin.
Therefore,
skin specimens are ideal for working
with anti -c yto ke rat in antibodies.
A brief o ve r v i e w of
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1
14
skin structure and the location of cytokeratin
be discussed.
in it will
Skin is composed of three di s t i n c t layers.
From the surface going d ee p they are: epidermis,
and subcutaneous fat
dermis,
(14).
The epidermis contains the greatest am oun t of cyto­
keratin.
It is made of a stratified squamous cornifying
epithelium, with cells called keratinocytes predominating.
The dermis is mostly connective tissue in w h ic h there are
blood vessels,
nerves,
eccrine sweat glands.
pilosebaceous-apocrine glands,
and
Below this is the s ub cutaneous fat
which varies in thickness according to location in the
body
(14,15).
The epidermis is compos ed of five regions of cells.
These are related in a developmental/cytomorphic gradient,
that is to say, the d eep est layers are immature forms of
the more superficial ones.
The outermost layer of corn-
ified cells is formed by the maturation of the epidermal
keratinocytes
superficial,
(14).
The epidermal layers,
from deep to
are:
1. Stratum g er min a t i v u m - cuboidal to columnar,
mitotically active cells.
2. Stratum spinosum - cells which contain elongated
nuclei,
and have cytoplasmic processes which att a c h to
adjacent cells by forming desmosomes.
3. Stratum gra nul os um - flattened cells w h i c h contain
conspicuous keratohyalin granules.
4. Stratum lucidum - closely compacted eosinophilic
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15
layer with no nuclei,
5.
found only in thick skin.
Stratum corneum - many layers of flat,
cornified,
anuclear cells with k er a t i n being the major c o mpo ne nt
(15).
The cell morphology and location of these regions are seen
in Figure 3.
It has been immunohistochemically d o c um ent ed that,
general,
in
cytokeratin proteins can be detected in all
regions of the epidermis and in some skin adenexa including
hair follicles,
(16,17).
sweat glands,
and ducts of sebaceous glands
There also seems to be an association of cy tok er­
atin protein with des mosomes
in the epidermal cells.
This
suggests that c yt oke rat in may be related to cellular
architecture
(17).
Therefore,
these are the specific areas
where there will be immunohistochemical positivity for
cytokeratin staining.
Immunoperoxidase Staining
Immunoperoxidase staining is completed through a
series of antigen-antibody reactions.
Through these,
gens can be mi cro scopically demonstrated in tissues.
a nt i­
The
value of this type of staining is becoming increasingly
more apparent.
Specifically,
the presence of c er t a i n
antigens in tissues a nd/ o r cells can lend insight into a
disease entity which may be present,
through cell morphology alone
but not apparent
(18,19,20,21,22).
The ultimate goal of this technique would be to help
insure proper disease diagnoses through the de t e c t i o n of
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16
* a r a t e t : S- corneum
S. lucidum
S. granulosum
S . spinosum
S - germinativum
B
Fig.
3. Cell layers of skin.
A. cell layers which reflect stages of keratinization - the vertically oriented columna r basal
cells are transformed into horizontally aligned
thin c orn ifi ed cells, taken from Moschella and
Hurley (20) .
B. section throu gh skin of human shoulder, taken
from Bloom and Fawcett (15).
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various antigens.
The pot e n t i a l
based in the literature
(20,21).
on which a diagnosis is based
tumor cells.
for this seems to be solidly
For instance,
one aspect
is the site of origin of
In cases of p o o r l y differentiated malignant
neoplasms the origin or p r i m a r y site of growth of cells may
be very difficult to establish.
Identification of specific
substances in these cells may help make this more clear.
For example,
the difference b e t w e e n poorly differentiated
lymphomas and undifferentiated carcinomas may not be apparent
through cell morphology.
But,
in the cells would indicate it
would,
the presence of cytokeratin
is of epithelial origin,
therefore be an e p i t h e l i a l l y derived carcinoma,
not a mesenchymally derived lymphoma.
antigen w hich can be detected
and
and
Cytokeratin is an
th rough immunoperoxidase
staining in all epithelial t i s s u e s
(23).
Peroxidase-Antiperoxidase
Staining for Cytokeratin
The peroxidase-an tip er oxi das e
(PAP) method of i m m u n o ­
peroxidase staining is an e x t r e m e l y sensitive and specific
technique.
Antibodies d i r e c t e d towards a variety of human
antigens including hormones,
globulins,
viruses,
proteins,
immu no­
and enzymes are c o m m e r c i a l l y available
They can be used on routinely
frozen sections,
In general,
smears,
(24).
fixed paraffin sections,
or c y t o s p i n preparations.
the procedure consists of a series of
antigen-antibody reactions,
in which an antibody molecule
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18
containing an enzyme is attached to antigenic sites
tissues.
Then a substrate solution is added.
enzyme and substrate react,
in
When the
a chromogen is formed,
and the
antigenic sites in the tissue become microscopically
visible through the presence of the color.
time,
At the same
the morphologic detail of the tissue section is p r e­
served.
The antibodies used
in the process are prepared
beforehand by the injection of antigens into laboratory
animals,
eg., rabbits or swine, which produce IgG molecules
against the specific antigens
(18).
The antibodies are
separated from the animal blood and are purified.
The PAP procedure,
(see p.
62)
as described in a DAKO PAP kit
is as follows;
1. Hydrogen peroxide is applied to the tissue for 5
minutes.
This reacts with any endogenous peroxidase present
in the tissue,
and will prevent it from reacting with the
substrate later.
This makes the procedure more specific
for the anti-cytokeratin antigen
2. Next,
(24).
serum from a normal swine
been given the antigen)
for 20 minutes.
(one which has not
is applied to the tissue sections
This reacts with the non-specific binding
sites in the tissue,
such as are found in collagen.
This
is necessary so the primary antibody in the next step does
not cross-react with the non-specific binding sites,
interpretation difficult
making
(24).
3. The primary antibody,
rabbit anti-cytokeratin,
applied to the sections for 30 minutes.
The antibody
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is
19
reacts and becomes chemically bo und to the antigenic binding
sites of cytokeratin
(24).
4. Swine anti-rabbit link serum is then placed on the
tissue for 30 minutes.
This combines with the primary
anti -cytokeratin already bound in the tissue.
It acts as a
link between the primary a n ti -cy to ker at in and the peroxidaseantiperoxidase, which comes in the next step.
This normal
serum must be from the same species as the link serum in
step 2.
Because antibodies have two binding sites, the link
is added in excess to ensure that at least one site on the
antibody remains free to bind the PAP
(24).
5. Peroxidase-antiperoxidase reagent is applied to the
tissue next for 30 minutes.
This a pe ntagonally shaped
molecule made of an antigen-a nti bod y complex.
it consists of three h o r s e r a d i s h
IgG molecules,
Specifically
peroxidase molecules and two
as is seen in Figure 4.
The peroxidase is a g lyc opr ot ein w it h an 18% carbo­
hydrate content.
Because this g lycoprotein portion is not
necessary for the enzymatic activity wit h the peroxidase,
it can be oxidized with p e r i oda te to form aldehyde groups.
It is these groups which are c ou ple d to the amino acid
groups of the IgG to form the PAP molecule
(18).
This
molecul e binds with the link antibody w h e n it is applied
to the tissue.
6. Lastly,
aminobenzadine
a substrate solution,
prepared with 3,3'-di-
(DAB), hydrog en peroxide,
and phosphate
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20
Fab
Fab
Fc
Fc
IgG
IgG
Fab
Fab
P = peroxidase molecule
F i g . 4. Schematic diagram of a peroxidase-antiperoxidase
molecule, taken from Sternberger (18).
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21
buffered saline
utes.
(PBS)
is placed on the tissue for 10 m i n ­
The peroxidase catalyzes the release of oxygen from
hydrogen peroxide, with subsequent oxidation of DAB.
results
This
in the localized precipitation of an insoluble
polymer that is brown
(the chromogen)
(20).
As can be seen
in Figure 5, wherever c yt oke rat in is present in a tissue
section,
a brown color will occur.
This enables one to
m icroscopically observe the presence of this antigen in
cells.
Rationale
The increasing widespread use of the immunoperoxidase
method of staining warrants the need for improving the
efficiency of the technique.
One of the major problems of
the procedure is the amount of time needed for its c o m ­
pletion.
If the immunoperoxidase procedure could be
carried out in less time than the several hours now r e ­
quired,
it would be an even more valuable tool for the p a t h ­
ologist.
This is the primary reason why this project will
attempt to utilize microwave radiation produced in a
m i crowave oven
to decrease the time required for the
immunoperoxidase reactions.
A n oth er reason deals with the value of this staining
procedure.
Immunoperoxidase staining can be used to help
identify the primary site or cell origin of tumor cells.
This information is critical
nosis,
for proper diagnosis,
prog­
and treatment of c ertain neoplastic diseases.
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22
BROWN
substrate
DAB
PAP
enzyme
SWAR
link
a nti-cyto­
keratin
primary
antibody
cytokeratin
antigen
A
Fig.
B
C
D
5. Schematic Dia gr am of Peroxiaase-Antiperoxidase
Procedure, adapted from Taylor (19).
A. application of primary antibody to antigen
B. ap plication of swine anti-rabbit link
C. appli ca tio n of PAP enzyme complex
D. c ompleted procedure with DAB added
KEY:
jtk
▲
^
cytokeratin antigen
primary antibody
swine anti-rabbit link antibody
peroxidase-antiperoxidase complex
BROWN
diaminobenzadine
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23
Therefore,
this p ro c e d u r e has great potential for use
in surgical pathology.
The practicality of the MW oven as a pi ece of laboratory
equipment is another reas on this project w o u l d be reason­
able to perform.
to purchase,
It is a relatively inexpensive instrument
and it is convienient to use and store.
For
these reasons the MW ov en would be readily ad a p t a b l e to a
laboratory setting.
The peroxidase-antiperoxidase method for cytokeratin
localization was c h o s e n as the standard for this project
for several reasons.
It has been well doc u m e n t e d in the
literature as giving g ood results by the routine method,
wh ich is attributed to the stability of c y t o k e r a t i n as an
antigen
(25,26).
Its antigenicity seems to w i t h s t a n d
histological processing very well.
cytokeratin proteins,
Skin is a b u n d a n t in
and is readily accessible in large
enough quantity to en su r e constancy for m ul tip le test
trials.
Because of all the above stated advantages of the
immunoperoxidase m e t h o d of staining for cy t o k e r a t i n in the
skin,
this project w i l l use the MW oven to a tt e m p t to
reduce the amount of time required for this procedure.
The overall reasoning behi nd this concept is b a s ed on the
premise that expediting this staining p rocedure may make
it a more useful m et h o d to detect medically impo rta nt
substances in tissue,
particularly those found in tumors.
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MA TER IAL S AND METHODS
Using sections of hu man thigh skin
(6 microns thick)
from randomly selected surgical specimens obtained at the
West Haven Veterans A d m i n i s t r a t i o n Medical Center,
peroxidase staining for c y t o k e r a t i n was done
A Sears Kenmore micr owa ve oven
immuno­
(see Plate 1) .
(model number 5658758410)
with an output of 650 w a t t s was used in an attempt to r e ­
duce the amount of time for staining.
using maximum MW power
This work was done
("Hi" setting on MW oven),
and v a r y ­
ing time only.
The quality of all slides was evaluated with respect
tointensity and
localiz ati on
of the color change produc ed
by the enzyme-substrate reaction.
Staining of the specific
areas believed to co nt a i n cytokeratin,
eg., epidermis,
were
graded on a scale of 0 to +4, with 0 representing no stain
reaction and +4 representing the most intense
Non-specific background areas,
in terms of none
overstained
(N), s l i g h t
(I.O.)
(see Plate 2).
eg., dermis, were evalu at ed
(S), moderate
(see Plate 3).
(M), or intensely
Using this system the
ideal MW exposure time wa s selected for each tested step.
Preliminary studies were done to determine the t o l e r ­
ance
of the skin sections and immunoperoxidase reagents to
MW exposure.
first,
Based on these,
the remaining studies tested,
the individual tol e r a n c e of each selected PAP
reagent;
and second,
the combi ned tolerance of all the
24
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Plate 1. Human skin from thigh stained by routine PAP
procedure for cytokeratin (see appendix I and II).
A. Negative control stained without anti- cytokeratin
(0 on grading scale) (40X).
B. positive control (+3 on grading scale)(40X).
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Plate 2. Illustration of grading scale used to evaluate
specific cytokeratin stain reaction in a PAP
procedure.
Human skin from thigh.
A. +1, B. +2, C. +3, D. +4 (40X).
For illustration of stain quality equal to 0,
refer to Plate 1.
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27
Plate 3. Illustration of grading scale used to evaluate non­
specific bac kground staining reactions in a PAP
procedure for c ytokeratin
(note the skin d e r m i s ) .
A. none (N), B. slight (S), C. moderate (M) and,
D. intensely overstained (I.O.). (40X).
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28
selected PAP reagents to acc um ula ted MW exposure in the
same PAP procedure.
swine serum
The s e le cte d reagents were:
(step 3), primary antibody
anti-rabbit link serum
(step 5),
normal
(step 4), swine
and PAP complex
(step 6).
These four reagents were c h o s e n for MW treatment b ecause
they require the longest incubations.
Testing individual serum r eag en t tolerance to M W e x p o ­
sure was done by adding brief M W exposures to an o t h erw ise
routine PAP procedure.
First,
normal serum was ap pli ed to
five skin sections at step 3 in a PAP procedure.
T hree of
these five slides were irra di ate d one at a time in the MW
oven.
The length of irradiation time, chosen according to
the preliminary tests, was 15,
removal from the MW oven,
20, and 25 seconds.
t hese three slides,
After
as w el l as the
two non-microwaved slides, w e r e continued through a routine
PAP procedure for cytokeratin.
The two slides not treated
in the MW oven were used as p os it ive and negative controls.
This same procedure was done three more times, once each
for:
anti-cytokeratin serum,
and PAP complex.
the 0 to +4 scale.
swine anti-rabbit link serum,
These 12 slides were graded according to
This d e t e r m i n e d if each of the four
reagents was affected in any w a y by the three MW exposu re
times as well as the optimum M W treatment time for eac h
of the four r e a g e n t s .
Using
these optimum MW e xpo sur e treatment times,
it
could be determined whether the immune reactions of these
four reagents could be com p l e t e d using MW treatment,
with
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29
no additional room temperature incubation.
of the four PAP serum reagents w as
again in the MW oven.
stained,
To do this e a c h
individually tested
Sections of skin were routinely PAP
except for the step at w h i c h a particular r e a g e n t
was to be tested.
First,
normal serum was tested.
Three slides of s k i n
wer e rout ine ly brought to step 3 in a routine PAP procedure.
Then two were continued through the process for use as p o s ­
itive and negative controls.
T h e one remaining slide was
irra diated in the MW oven wit h n orm al swine serum on it for
the o p t i m u m time chosen from pre v i o u s wo r k
seconds).
(15, 20, or 25
No additional t r e a t m e n t at room temperature w a s
done after this.
Instead,
the slide was immediately c o n ­
tinued through the rest of the PAP procedure.
showed positive results,
If this slide
it w o u l d be known that the n o r m a l
serum reacted properly while u n d e r M W treatment,
shorter du ration than is rou t i n e l y used.
but for
Similar te ch niques
were used to individually test the reactivity of the p r i m a r y
antibody,
the swine an ti-rabbit link serum, and the PAP
complex.
The next part of the p r o j e c t was to perform a c o m p l e t e
PAP procedure using MW t re at men t at all of the four se lec t e d
steps.
A PAP procedure was done,
appropriately s u b s t i t u t ­
ing M W treatment for routine r o o m temperature tr eatment a t
the p ro per point in the p r oce du re for all four of the
involved reagents.
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The established procedure of reduced time was then
used to stain five surgical cases which were routinely
determined to be cytokeratin positive by staff pathologists
at the West Haven VA Medical Center.
Also, one standard
positive case, already shown to give good quality MW s tai n­
ing results,
and one case known to be cytokeratin negative
were stained by the same MW procedure for use as positive
and negative controls,
respectively.
All sections were
then evaluated for quality using the previously described
grading systems.
These were also c om par ed to corresponding
routinely PAP stained sections.
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RESULTS
Effects of Microwave Treatment on Individual
PAP Serum Reagents
Physical Tolerance of Tissue Sections
and PAP Serum Reagents
to Microwave Exposure
These tests determined that 1 . 3 - 5
drops of water on
a slide in a moist chamber would not evaporate un de r 650
watts MW exposure for up to 45 seconds;
2. normal swine
serum, used as a representative for all the PAP reagents,
could withstand this degree of MW exposure for up to 30
seconds before coagulation of the serum occurred/
tissue morphology,
and 3.
eval uat ed after routine H&E staining, was
not altered by a 30 second MW exposure of serum cov er ed tis­
sue sections in a moist chamber.
Individual Microwave Irradiation of
Serum PAP Reagents with Subsequent
Routine Incubations
After 15, 20, or 25 seconds M W exposure,
plus
sequent routine incubation at room temperature,
the sub­
all four
PAP reagents retained their abilities to function properly
(see Table 3).
This was
indicated by positive PAP staining
for cytokeratin which was of equal or better q u a l it y than
the non-microwaved control.
In general,
the longer MW exposures produced positive
PAP staining of better qual ity than the shorter exposures.
31
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32
Table 3
Individual Microw av e Irradiation of S e r u m
PAP Reagents with Subsequent
Routine Incubations
Reagent
MW exposure
time (sec)*.
Resulting s t a i n
r e a c t i o n s ♦♦
cytokeratin
ba c k g r o u n d
15
+1
N
20
+1
N
25
+2
N
15
+3
N
20
+4
N
25
-t-3
N
15
+2
N
20
+2
N
25
+3
N
15
+2
N
peroxidase-anti­
peroxidase serum
20
+2
N
25
+3
N
positive control
0
+3
N
negative control
0
0
N
normal swine serum
anti-cytokeratin
serum
swine anti-rabbit
link serum
♦Immediately after the indicated MW exposures, the sections
were left to incubate at room temperature as follows:
normal swine serum - 20 min., anti-cytokeratin - 30 min.,
swine anti-rabbit link serum - 30 m i n . , and PAP c o m p l e x 30 min.
♦♦Results for specific areas believed to contain cyt o k e r a t i n
are reported on a scale of 0 to +4, with 0 re pr ese n t i n g
no stain reaction and +4 representing the most intense
staining.
Results for non-specific background areas of t i s s u e are
reported in terms of none (N) , slight (S) , m o d e r a t e (M) ,
or intensely o verstained (I.O.).
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1
33
MW treatment of the primary antibody significantly enhanced
the intensity of PAP staining of cytokeratin-containing
cells,
without significant increase of non-specific b a c k ­
ground staining in any of the sections
(see Plate 4).
Microwave Irradiation of PAP Serum
Reagents Without Routine
Incubations
Individual testing of each PAP serum reagent for its
proper function after the brief MW treatment times revealed
that the normal swine serum step and the swine anti-rabbit
link serum could be successfully completed in the MW oven
in 25 seconds
(see Table 4).
serum step required 30 seconds
primary antibody step,
The peroxidase-antiperoxidase
(see Tables 4 and 5) .
The
after several different variations
of brief MW treatment, was shown to give the best results
by an initial 20 second MW exposure,
immediately followed
by incubation for 30 minutes at room temperature.
The
various methods used to shorten this step and a descrip tio n
of the results are outlined in Table 6.
Effects of M icrowave Treatment on Combinations
of PAP S eru m Reagent Steps
Co mbination of Microwave Treatments Which
Were Shown to Give Positive Results
Individually
The steps which were ex pediated by MW treatment
Tables 3,4,5)
were combined in one procedure
(see Table
Three separate trials of the procedure were run.
sections showed positivity
(see
The
in the appropriate cells of
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7) .
34
Plate 4
Com par is on of routine and micr owa ve PAP procedure
for cytoker ati n stain reactions.
Human skin from
thigh.
A. stained by routine PAP procedure (+3 reaction) .
(40X).
B. stained by adding a 20 second MW exposure to
only the anti-cytokeratin step in a PAP procedure
(+4 reaction, as indicated in T able 3) (40X).
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35
Table 4
MICROWAVE IRRADIATION OF PAP SERUM REAGENTS
WI TH O U T ROUTINE INCUBATIONS
Reagent
MW exposure
time (s e c )♦
Resulting stain
re a c t i o n ^ ♦
cytokeratin
background
normal swine
serum
25
+3
N
anticytokeratin
serum
20
+1
N
swine anti-rabbit
link serum
25
+3
N
25
+2
N
PAP complex
♦The three reagents not being tested at a pa rt icular step
were routinely used in the PAP procedure as follows:
normal swine serum - 20 min., anti-cytokeratin se rum - 30
min., swine anti-rabbit link serum - 30 min., and PAP
complex - 3 0 min. at room temperature.
♦♦Results for specific areas believed to contain cytokeratin
are reported on a scale of 0 to +4, with 0 represe nt ing no
stain reaction and +4 representing the most intense s t ai n­
ing .
Results for non-specific background areas of tissue are
reported in terms of none (N), slight (S), m od era te (M),
or intensely overstained (I.O.).
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36
Table 5
FURTHER MI C R O W A V E TESTING OF ANTI-CYTOKERATIN
AND PEROXIDASE-ANTIPEROXIDASE
REAGENTS
Reagent
MW exposure
time (sec)*
Resulting stain
reaction* *
cytokeratin
anti-cyokeratin
serum
peroxidaseantiperoxidase
serum
background
N
30
+1
40
+4
1 .0.
50
+4
1 .0.
30
+3
N
40
0
N
50
0
N
*Except for the reagent being tested, all ot her steps in the
PAP procedure were done routinely as follows: normal swine
serum - 20 min. at room temp., anti-cytokeratin serum - 30
min. at room temp., swine anti-rabbit link serum - 30 min.
at room temp., and PAP complex - 30 min. at room temp.
**Results for specific areas believed to contain cytokeratin
are reported on a scale of 0 to +4, with 0 representing
no stain reaction and +4 representing the most intense
staining.
Results for n on -specific background areas of tissue are
reported in terms of none (N), slight (S), moderate (M),
or intensely ov e r s t a i n e d (I.O.).
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37
Table 6
METHODS USED TO REDUCE REACTION TIME OF
ANTI-CYTOKERATIN REAGENT
Resulting stain
reaction^♦
Method used to shorten anticytokeratin step^
cytokeratin
background
+1
N
30 sec. MW exposure
+1
N
4 0 sec. MW exposure
+3
M
50 sec. MW exposure
+3
M
30 sec. MW exposure, 5 min.
room temp, incubation, 30 sec.
MW exposure
+4
30 sec. MW exposure, 10 min.
room temp, incubation, 30 sec.
MW exposure
+4
W
•
20 sec. MW exposure
30 sec. MW exposure, 15 min.
room temp, incubation, 30 sec.
MW exposure
+4
1 .0.
•
O
I .O.
*All other steps in the procedure are carried out by the
routine method, i e ., no MW exposure.
♦♦Results for specific areas believed to contain cytokeratin
are reported on a scale of 0 to +4, with 0 representing
no stain reaction and +4 representing the most intense
staining.
Results for non-specific background areas of t i s ­
sue are reported in terms of none (N), slight (S), moderate
(M), or intensely overstained (I.O.).
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38
Table
7
COMBINED MICROWAVE TR E A T M E N T PAP PROCEDURE
Reagent
normal swine serum
anti-cytokeratin
serum
swine anti-rabbit
link serum
peroxidase-antiperoxidase serum
R e action procedure*
25 sec in MW oven
20 sec in MW oven plus
30 min at room temp
25 sec in MW oven
30 sec in MW oven
This was done using exposure times obtained from the opti mu m
results of the previous tests.
*In this procedure the above steps are carried out in the
proper sequence to complete a PAP procedure.
No steps
are done by the routine method.
Between each step slides
are w ashed in PBS buffer for 10 minutes at room temperature.
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39
intensity from + 2 or +3.
However,
there was a slight to
moderate increase in non-specific background staining.
This was not as intense as in the positively stained cytokeratin-containing cells.
The negative controls which
utilized normal serum at the primary antibody step,
dis­
played evenly distrib ut ed slight to moderate n on -specific
staining over the entire section in each of these trials.
These particular sections were also MW treated.
The cyto-
keratin sites did not stain more intensely than the b a c k ­
ground in these same sections
(see Table 8).
Microwave Treatment of All PAP
Serum Reagents Except the
Primary Antibody
When MW exposure of the anti-cytokeratin serum was
omitted
(see Table 9), all cells containing cyt oke ra tin
displayed a positive reaction of +3 intensity.
background staining was still present,
degree.
Althoug h
it was to a lesser
Two of the negative control slides,
also M W treated,
displayed a similar pattern of non-specific staining over
the entire section,
Table 10).
as in the three previous trials
(see
Trial 2 gave the best results which are
illustrated in Plate 5.
Microwave Staining of Five Surgical
Specimens Containing C ytokeratin
Five surgical cases,
previously determined to show
good quality PAP reactions for cytokeratin by the routine
method, were stained using MW treatment during all
serum
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40
Table 8
RESULTS OBT A I N E D USING COMBINED MICROWAVE TR E A T M E N T
FOR ALL PAP SERUM REAGENTS *
Trial
Serum used at pri­
mary ant i b o d y step
Resulting stain
reaction^ *
cytokeratin
1
immune
negative control
immune
background
+3
S
N/A
S
+2
M
N/A
M
+3
M
N/A
M
2
negative control
immune
3
negative control
♦The procedure used in all three trials is: normal swine
serum - 25 sec in MW oven, anti-cytokeratin s erum - 20
sec in MW oven immediately followed by 30 min room temp
incubation, swine anti-rabbit link serum - 25 sec in MW
oven, and PAP c o m p l e x - 30 sec in MW oven (see Table 7).
♦♦Results for specific areas believed to contain c y t o k e r ­
atin are reported on a scale of 0 to +4, with 0 repre­
senting no stain reaction and +4 representing the most
intense staining.
N/A = nonapplicable because both specific and non-specific
areas of tissue stained the same.
Results for non-specific background areas of tissue are
reported in terms of none (N), slight (S), m o der at e (M),
or intensely overst ain ed (I.O.).
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41
Table 9
COMBINED MICROW AV E TREATMENT PAP PROCEDURE*
FOR AL L STEPS EXCEPT THE
PRIMARY ANTIBOD Y STEP
Reagent
normal swine serum
Reaction procedure*
25 sec in MW oven
anti-cytokeratin serum
30 min at room temp
swine anti-rabbit
link serum
25 sec in MW oven
peroxidase-antiperoxidase serum
30 sec in MW oven
♦This procedure is modifie d from the initial procedure
(Table 7) used to attempt a reduction of non-specific
background staining.
♦♦The above steps are ca rr ied out in proper sequence to
complete a PAP procedure.
Between each step slides are
wash e d in PBS buffer for 10 min at room temperature.
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42
Table 10
RESULTS OBTAINED USING COMBINED MICROWAVE TREATMENT* FOR
ALL PAP REAGENTS EXCEPT ANTI-CYTOKERATIN
■
Trial
Serum used at p r i ­
mary antibody step
■
■
■
■ —
cytokeratin
immune
2
immune
negative control
immune
■
background
+3
S
N/A
S
+3
N
N/A
N
-*-3
M
N/A
M
1
negative control
~
Resulting stain
reaction* *
3
negative control
*This procedure, used for all three trials, is: normal swine
serum - 25 sec in MW oven, anti-cytokeratin serum - 30 min
at room temp, swine anti-rabbit link serum - 25 sec in MW
oven, and PAP complex - 30 sec in MW oven (see Table 9).
**Results for specific areas believed to contain c ytokeratin
are reported on a scale of 0 to +4, with 0 representing
no stain reaction and +4 representing the most intense
staining.
N/A = nonapplicable because both specific and non-specific
areas of the tissue stained the same.
Results for non-specific areas of the tissue are reported
in terms of none (N) , slight (S), moderate (M), or
intensely overstained (I.O.).
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43
Plate 5. Comparison of routine PAP procedure reaction and
successful microwave exposed PAP procedure reaction.
Human skin from thigh.
A. stained by routine PAP procedure (+3 react ion ).
(4 0 X ) .
B. stained by using MW treatment only for all steps
except ant i-cytokeratin (see Table 9 for this
procedure; also a +3 reaction) (40X).
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applications except that of the primary a nt ib ody
9).
One negative control
specimen
to be cytokeratin negative)
(see Table
(previously determined
and the standard s k i n positive
control were also stained in this manner.
The PAP MW
reaction results were similar for each case t e s t e d
Table 11).
In general,
positive staining,
(see
the procedure produced good quality
but non-specific background
present in most sections.
staining was
In three of the five test cases
(A , B ,C ) the non-specific background staining w a s not as
intense as the specific
in two cases
(D,E)
staining of cytokeratin.
However,
no diff er enc e in intensity w a s seen
between specific and non-specific staining.
When normal serum
case,
(negative control)
was u sed
two of the six c ases had no non-specific
for each
staining.
The four remaining cases showed slight to mo d e r a t e overall
staining.
In two of these four cases there was
no d i f ­
ference between staining of sections on which the primary
antibody serum
(anti-cytokeratin) was used,
and secrions on
which normal serum was used in place of anti-cytokeratin.
Both the negative c ontrol sections,
one stained with anti-
cytokeratin serum and one stained with normal
not have shown any positivity;
tain cytokeratin.
However,
serum,
should
because they did not c o n ­
there was evenly di s t r i b u t e d
non-specific staining o v e r the entire section in both of
these,
as is shown in T a b l e 11.
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45
Table 11
COMPARISON OF ROUTINE AND MICROWAVE
STAINING OF SPECIMENS KNOWN
TO C O N T A I N CYTOKERATIN*
Routine PAP reaction results
Case
positive
immune serum
MW PAP reaction results
negative
control
serum
positive
immune serum
negative
control
serum
CK & BG
CK
BG
CK & BG
CK**
BG**
A
+3
N
N
+2
S
S
B
+3
N
N
+3
S
N
C
+3
N
N
+3
S
S
D
+3
N
N
N/A
M
M
E
+3
N
N
N/A
M
M
positive
control
negative
control
+3
N
N
+3
N
N
0
N
N
N/A
S
S
♦Determined by staff pathologists at the West Haven VA
Medical Center.
**CK = cytokeratin and BG = background
Results for specific areas believed to contain c y t o k era tin
are reported on a scale of 0 to +4, with 0 representing
no stain reaction and +4 representing the most intense
staining.
N/A = nonapplicable because both specific and non-specific
areas of the tissue stained the same.
Results for non-specific areas of the tissue are reported
in terms of none (N), slight (S), moderate (M), or
intensely overstained (I.O.).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
D IS C U S S IO N
Data Analysis
The purpose of this project was utilization of the
microwave oven to improve the immunoperoxidase staining
technique.
This was to be accomplished by decreasing the
amount of time necessary to complete the procedure without
sacrificing the quality of the end product.
First,
normal serum on a section of skin from the
thigh was tested for MW tolerance.
This established a
maximum exposure time of approximately 30 seconds.
This
determination was based on whether the serum dried on the
slide as well as whether the microscopic morphology of the
e x posed skin sections remained unchanged.
Microscopic
ex am ina tio n of H&E stained exposed skin sections verified
the latter.
Normal serum was chosen as a representative
of the four PAP reagents because they are all serum based.
The only difference between reagents is that each contains
d i f fer en t specific antibodies.
Based on the time established above,
the functional
tolerance to MW exposure of all four PAP serum reagents was
tested next.
Each reagent was MW treated separately,
and
then allowed to incubate at room temperature for the
routine time indicated in Appendix
II.
The quality of
staining in each irradiated tissue section indicated if the
46
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47
particular reagent being tested functioned properly.
Table 3 indicates that at least one section tested for each
reagent produced good quality staining,
tivity was not destroyed.
thus reagent reac­
The successful MW exposure times
for all the PAP reagents were:
20 seconds for an ti-cytoker­
atin serum, and 25 seconds for normal serum,
rabbit link serum,
swine a n t i ­
and peroxidase-antiperoxidase serum.
These findings are important in two ways.
First,
it
has been conclusively determined that for the MW exposures
listed above,
MW radiation has no harmful effect on
reagents or tissues.
tinued.
Second,
Thus,
further testing can be c o n ­
as is indicated in Table 3 and is seen in
Plate 4, the 20 second MW exposure of anti-cytokeratin,
prior to the routine 30 minute incubation at room t e m p ­
erature,
greatly enhances the stain quality.
The intensity of staining in this particular skin
section was even much greater than in the positive n o n ­
microwaved control.
It is important to note the stain
positivity is localized and limited to those structures
in the skin which are believed to contain cytokeratin,
the epidermis.
ing.
eg.,
There is no non-specific background stain­
So, for this particular serum step, the added 20
second MW exposure improves the immunoperoxidase stain
quality.
This enhanced qua lit y staining is probably related to
the heat produced by the microwaves during irradiation
treatment.
When the slides with the serum were removed
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48
from the MW oven after each trial,
were warm.
it was noted that they
It has been long established that the rate of
most chemical reactions
increases as temperature rises
(27).
One may speculate that the heat produced by the MW oven
during the 20 second M W exposure increased the moveme nt of
the anti-cytokeratin molecules.
This
increases their
kinetic energy and allows them to bind to more antigenic
sites in the tissue, w h i c h results in more intense staining.
The question arises here whether the radiation from
the MW oven produces he at which then leads to increased
kinetic energy of the irradiated molecules,
kinetic energy increases
production.
or w hether
first, which then leads to heat
Because the PAP stain reaction occurring
after a 20 second MW ex po s u r e of anti-cytokeratin serum
was appropriately locali ze d in the tissue sections,
there
is no reason to believe that the antibodies or antigens
have been damaged,
as m i g h t be induced by heat.
Therefore,
it may be surmised that the MW irradiation caused increased
molecular kinetics, which,
rate.
in turn,
increased the reaction
This led to i ncreased stain intensity.
of heat produced,
then,
The majority
probably came after increased
kinetic energy.
The results ob ta i n e d in the next part of the project
(outlined in Tables 4 an d 5) determined that the PAP serum
reactions, except the an ti-cytokeratin step, could indeed
be completed with MW exposure.
This was concluded because
except for the step at w hich a particular reagent was being
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49
tested,
the remainder of the procedure was done routinely.
A positively stained section meant that all reactions
procedure were properly completed.
in the
It is likely that in­
creased molecular movement along with heat induced by the
microwaves are responsible for the reaction completeness
which occurred at each serum reaction step.
This allowed
for sufficient binding of antibody to antigen.
By the end
of the procedure there must be sufficient amount of p e r o x ­
idase
enzyme present on the tissue to react with enough
DAB substrate to give good quality staining.
The positive
staining is localized at the proper sites ir. the tissue,
where cytokeratin is be li e v e d to be present.
ie.,
Thus far the
procedure has not lost its specificity as a result of MW
exposure.
As has been mentioned,
the anti-cytokeratin step does
not appear to be co mpleted using MW exposures
4,5,6).
(see Tables
For MW exposures of 30 seconds or less,
ing quality of tissue sections resulted.
weak and focally d istributed
poor stain­
The staining was
(see Table 6).
These results
could be interpreted to mean the reaction was not completed
due to insufficient molec ula r agitation produced by the
microwaves.
It is not believed that the incomplete reaction of the
anti-cytokeratin step is due to destruction of the a n t i ­
bodies in the reagent or the antigens in the skin by the
microwaves.
This is because this reaction was
successfully
completed when sections and serum were irradiated for 25
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50
seconds,
minutes
then left to incubate at room temperature for 30
(see Table 3).
Here,
the MW exposure did not
result in the destruction of any necessary components of
the reaction.
For MW exposures of greater than 30 seconds, whether
as a continuous MW exposure or alternating with room t em p­
erature incubations, poor staining guality occurs.
In
comparison to a 30 second or less MW exposure, which re­
sulted in focal light staining,
these longer MW exposures
resulted in intense n on- specific overstaining.
Alternating
MW exposures with room temperature incubations should have
kept heat production to a minimim.
removal from the MW oven,
But, even after
increased kinetic energy of the
substances on the slide allowed the excess heat to remain.
The majority of heat produced by the microwaves,
then,
most likely occurs after kinetic energy has been increased.
Here,
30 seconds seems to be the critical point at which
heat becomes excessive.
It is believed that heat is re­
sponsible for this intense non-specific overstaining by
altering molecular c onf ormation of the exposed proteins.
Specifically,
the non-specific overstaining may pos­
sibly indicate that the binding sites on either the
antigen or the antibody are altered in such a way that the
primary antibody attaches to many non-specific binding
sites over the entire tissue section,
That is, protein structure,
not just cytokeratin.
probably molecular conformation,
may be altered by the MW induced heat.
This may occur on
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51
non-specific tissue proteins,
thus destroying the tissue
and allowing the Fc p ortion of the anti-cytokeratin to
bind anywhere.
On the other hand, the structure of the Fc
site itself may be altered,
Additionally,
thereby making it non-specific.
increased MW radiation continues to increase
molecular movement.
The combination of altered molecular
structure and increased molecular activity probably causes
the excessive non-specific overstaining which has been
observed.
At this point it was concluded that the reaction times
for only the normal serum,
swine anti-rabbit link serum,
and peroxidase-antiperoxidase serum may be possibly reduced
in one PAP procedure.
The primary antibody reaction would
best be done by a 20 second MW exposure followed by a 30
minute room temperature incubation.
This would still
greatly reduce the am ou n t of time required for the entire
PAP procedure.
This s h o rt ene d procedure is outlined in
Table 7.
The stain reactions following this procedure
(see
Table 8) were not of equal quality to slides stained by
the routine method.
positively,
A l t h o u g h the cytokeratin did stain
more non-specific background staining than was
present in the routinely stained control sections was also
present.
This may m a ke
interpretation of an unknown
tissue specimen uncertain.
As would be expected,
the negative control sections,
which were also MW trea ted and stained using normal
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52
non-immune serum in place of anti-cytokeratin serum,
the same non-specific staining.
In these sections,
showed
though,
cytokeratin was not more intensely stained than the b a c k ­
ground.
This means that the DAB was able to bind to the
tissue even though no anti-cyt oke ra tin was present.
It is believed that the mec han ism for the non-specific
staining observed is probably due in part to the c o n s e ­
quences of increased kinetic energy and heat production
which have already been discu sse d
(see pages 50-51).
But,
because each reagent was irradiated for only 30 seconds or
less;
and,
each slide was im mediately cooled in a room
temperature PBS bath,
increased kinetic energy and heat
can not be the only factors involved.
Therefore, c o m b i n ­
ations of individually successful MW exposures done in one
PAP procedure must lead to an accumulation of radiation for
each section stained.
This c u m u la ti ve effect of MW rad­
iation must go beyond molecular agitation and heat p r o ­
duction.
Some other unknown factor could be at blame for
the non-specific staining.
The non-specific staining could occur at any point in
the procedure.
It is probably the swine anti-rabbit link
serum or the peroxidase-antiperoxidase serum, or both,
which non-specifically bind to the tissue.
occur later in the procedure,
The normal swine serum step
after 25 seconds MW exposure
These steps
after accumulated MW exposure.
(step 1)
functioned properly
(see Table 4).
Therefore,
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53
the non-specificity is not due to unbound endogenous non­
specific binding sites in the tissue.
Endogenous tissue enzymes could be responsible for
some of the non-specific staining observed.
In the PAP
procedure hydrogen peroxide reacts with endogenous tissue
peroxidase
(see Appendix II).
This eliminates an inap­
propriate reaction with DAB later.
The accumulation of MW
radiation may increase the activity of endogenous peroxidase
left unbound by hydrogen peroxide.
The radiation may also
change the structure of other endogenous enzymes,
rendering
them capable of a reaction with DAB.
Next, a combined MW procedure,
with no MW exposure at
the primary antibody step, was done to attempt elimination
of the non-specific staining
(see Table 9).
This, however,
yielded results similar to those previously obtained
Table 10).
(see
The non-specific staining in the positive and
negative controls was of slightly lesser intensity, or in
one case,
not present at all
(see Plate 5).
The 20 second
decrease in cumulative MW exposure most likely accounts
for this.
Reproducibility of the above results was tested using
five surgical cases known to contain cytokeratin.
stain reactions
The
(see Table 11) were inconsistent and not
of diagnostic quality.
Generally,
not enough of a di f­
ference between slides stained with immune serum and those
stained with normal, non-immune serum was evident.
The
amount of cumulative MW radiation used here appeared to be
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54
incompatible with succe ssf ul completion of the immunoperoxidase reactions.
Microwave r ad i a t i o n can be used to im pro ve the immunoperoxidase technique;
not by decreasing the am ou n t of time
required for the procedure,
intensity.
but by enha nci ng the staining
In this w a y limited MW exposure improves the
quality of obtainable results
acity,
(see Plate 4).
In this cap­
the goal of u t i l i z i n g the MW oven to improve the PAP
technique has been met.
Although this is the most positive
significant finding r es u l t i n g from this project,
all the
data holds its own pa r t i c u l a r value.
The enhanced q u a l i t y of staining increases the ease with
wh ich immunoperoxidase stained slides are evaluated.
This
makes the technique mo re valuable as a di a g n o s t i c as well as
a research tool for pathology.
Also, the M W exposu re may
accentuate otherwise undetectable specific posi tiv it y on
tissues.
In these ways,
the results of this pr oject can be
applied to the general field of pathology.
The remaining d a t a holds most significance for future
w o r k on this topic.
The fact that each in dividual PAP serum
reagent is not des t r o y e d by a limited amo unt of MW r a d ­
iation,
is of p ar ticular importance.
This
i nformation
implies there may still be a way to use the M W o ven to
decrease the time r e qui re d for immunoperoxidase staining.
As long as the specific reactivity of the a nt ige ns and
antibodies can be maintained,
work towards
this goal can
be continued.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
It would be worthwhile to continue the efforts ocgun
here to shorten the PAP procedure.
The first suggestion
for follow up work on this project is to develop a way to
monitor the temperature of the reagents during MW treatment
This capability would allow for the use of lower MW setting
In this way the antigens and antibodies could be exposed to
a lesser degree of radiation.
The exposure,
however,
might
be enough to successfully complete the required immune
reactions.
Determination of the exact point at which MW radiation
becomes excessive is another suggestion for follow up work.
If this point at wTiich the tissues and/or the reagents b e ­
come destroyed was known,
possioly still be used,
three steps.
a comoined MW7 procedure could
even if only for the first two or
The total procedure time could still be si g­
nificantly reduced.
Another approach may separate the increased reactivity
from the detrimental loss of specificity.
were Kept cool,
If the slides
possibly on ice, during the MW exposure,
denaturation of proteins may be reduced.
This is assuming
that the non-specificity was a result of heat and not a
direct effect of microwaves.
Elimination of non-specific background staining would
possibly improve the q uality of the stain reaction results
of a PAP MW procedure.
as trypsin and pepsin,
Recently,
proteolytic enzymes,
have been successfully used in
routine PAP procedures for this purpose
(27,28).
It has
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such
56
been found that such enzyme treatment
antigens,
thereby making
"unmasks" tissue
immunohistochemical reactions more
specfic by reducing non-specific background
staining.
The
mechanism by which tnis occurs is thought to be related to
enzymatic activity which
1) destroys ground substance pro­
tein molecules that are in connective tissue
(27), or 2)in-
creases cell and tissue permeability to the macromolecules
that are in the PAP reagents
sibility of the antigens
treatment,
(28).
This increases acces­
in the cells.
Such an enzyme
then, wo uld be worthwhile to try when performing
a MW PAP p r o c e d u r e .
Overall Conclusions
The goal of this project was to dev e l o p an immunoper­
oxidase staining technique which implements
a MW oven, and
requires less time than is routinely necessary.
Several
preliminary tests we re performed in order to provide the
basic information required to formulate the final method
which was developed
(see Table 9).
These tests involved
using the MW oven in several PAP procedures with careful
evaluation of each slide stained.
The conclusions drawn from the sequence of tests done
in this project are:
1.
Individual MW irradiation of skin sections covered
with PAP antibody and enzyme reagents for 30 seconds or
less does not destroy the reactivity of antigens or anti­
bodies required for good quality PAP staining.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
J /
2. Cumulative MW expos ure s
in one PAP procedure Joes
appear to destroy the involved PAP reagents or the tissues,
or both,
resulting
in non-specific staining along with
increased intensity.
3. The probable cause of the unfavorable staining
results is related to the increased Kinetic energ y of the
exposed substances,
and the subsequent heat production,
as well as possible u nk n o w n factors.
4. The MW oven can be used to greatly e nhance tire stain
intensity resulting f r om a routine PAP procedure for cyto­
keratin.
This is done by a 20 second MW exposure of the
tissue section covered with the anti-cytokeratin serum
prior to routine room temperature incupation.
Increased
molecular movement duri ng this brief MW exposure leads to
increased stain intensity without loss of specificity.
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DeLellis R . , S ternberger L . , Mann L . , Banks P., Nakane
P: Immunoperoxidase Techniques in Diagnostic Pathology.
Report of a Works ho p Sponsored by the Nat:onal Cancer
Institute. Am. J. Clin. Pathol. 7_1, 483-488 (1979).
22. Banks P.: Diagnostic Application of an Immunoperoxidase
Method in H e m a t o p a t h o l o g y . J. Histochem. Cytochem. 2 7 ,
1192-1194 (1979) .
23. Nagle R., McDaniel K., Clark V. , Payne C.: The Use cf
Antikeratin Ant ibodies in the Diagnosis of Human N e o ­
plasms Am. J. Clin. Pathol. 79_, 458-466 (1983) .
24. DAKO PAP Kit Instruction Booklet. DAKO Corporation,
22 North Milpas St., Santa Barbara, CA 93103 (1980).
25. Gabbiani G, Kapanci Y., Barazzone P., Werner F.:
Immunochemical Identification of Intermediate-sized
Filaments in Human Neoplastic Cells. Am. J. Pathol.
104, 206-216 (1981) .
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
60
26. Schlegal R., Schlegal S., McLeod J., Pinkus G.: Immuno­
peroxidase Local iza tio n of Keratin in Human Neoplasms.
Am. J. Pathol. 101, 41-50 (1980).
27. Reading M . : A Diges tio n Technique for the Reduction of
Background Staining in the Immunoperoxidase Method.
J. Clin. Pathol. 3_0' 88-90 (1977).
28. Curran R. and Gregory J. : Demonstration of Immuno­
globulin in Cryos ta t and Paraffin Sections of Human
Tonsil by Immunofluoresence and Immunoperoxidase
Techniques. J. Clin. Pathol. 3_1, 974-983 (1978).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
A P P E N D IX
I
Reagents Used in the PeroxidaseA n t i p ero xi das e Procedure*
1•
3% H 2 O 2
2.
Normal swine serum
3.
Primary antibody
4.
Link antibody
5.
PAP complex
6.
Non-immune rabbit serum
7.
DAB reagent: Add 2-3mg DAB to 2.5ml PBS,
before use, add 5-8 d rops 3% H 2 O 2
8.
Xylene
9.
Ethanol;
10.
Phosphate buffered saline
11. Harris'
100%,
95%,
(negative control)
immediately
80%
hematoxylin
12. Ammonia water: mix 15ml of c o n e . ammonium hydroxide
with 1 liter tap water
13. Acid alcohol:
alcohol
mix 10ml c o n e . HCl with 1 liter 70%
14. EUKITT mounting me di um (Calibrated Instruments,
731 Saw Mill River R d . Ardsale, New York 10502)
♦Reagents 1-7 obtained from DAKO Corporation,
Milpas St. Santa Barbara, CA 93103.
Inc.
22 North
Reagents 8-14 routinely used at the West Haven V A Medica
Center, West Haven, CT 06516.
61
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
A P P E N D IX
I I
Pero xi das e-Antiperoxidase Staining Procedure*
1. If paraffin sections are used,
deparaffinize sections in 2 xylene baths.
R e h ydr at e in graded alcohols, 100%, 95%,
and 80%.
2. Wipe slides around tissue a n d place in
mo ist chamber.
Apply 2-3 drops 3% H 2 O 2
to cover tissue, incubate.
Wash slides in Phosphate b u f f e r e d saline (PBS)
5 min ea.
3 min ea.
5 min
10 min
3. Place slides in moist chamber, apply normal
serum to specimen and incubate.
20
min
4. Tap off excess serum and a p p l y primary
antibody to s e c t i o n s , use non-immune
serum as negative control, incubate.
Wash slides in PBS.
30
10
mm
min
5. Wipe slides around tissue.
Apply link
antibody to tissue sectio ns and incubate.
Wash slides in PBS.
30
10
min
min
6. Wipe slides around tissue,
reagent, incubate.
Wash slides in PBS.
Apply PAP
30 min
10 min
7. Place slides in moist c h a m b e r and apply
freshly prepared D i a m i n o b e n z a d i n e - H 2 0 2
r e agent to slides.
Rinse slides in PBS.
10 min
10 min
8. Cou nterstain slides in Harris' hematoxylin.
Wash in tap water.
Rinse briefly in acid alcohol.
Place slides in ammonia water.
Wash in tap water.
Dehydrate slides in g r a d e d alcohols: 80%
95%
3 baths 100%
Place slides in two xyle ne baths.
5 min
5 min
2 dips
till blue
5 min
2 dips
2 dips
2 dips ea,
2 min ea.
9. Cove rs lip with EUKITT
♦Procedure used at the West H a v e n VA Medical Cente r
62
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