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Observations on the histochemical reduction of photographic emulsion in radioautography.

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OBSERVATIOXS ON T H E HISTOCHERIICAL
REDUCTION O F PHOTOGRAPHIC EMULSION I X
RADIOAUTOGRAPHY
S E W T O N B. EVERETT AND BARBARA S. SIMMONS
The Ilepal-tment of Baatolit!/, University of Washmgtoii, GeatlEe
FOUR FIGURES
Recent attempts within this laboratory to demoiistratc by
radioautography the specific localization of low concentrations
of i.adioisotopes in rat tissues have emphasized the danger of
liistochemical fogging of photographic emulsion. Boyd and
Board ( '49) called attention to this phenomenon, and more
recently Board ( '51) implicated the tissue sulfhydryl groups
as responsible agents for this cllemical reduetion. Several investigators have considered the likelihood of misinterpretatioii
of tissue radioautographs due to histochemical fogging, and
have proposed methods to prevent this phenomenon, such as
the coating of tissue sections with a plastic film: for example,
by celloidin, Geoii Resin, o r Formvar (Gross et al., '51 ; Eidinoff et al., '51). Williams ( '51) developed a dry method of
inouiitiiig tissues, which was reported to prevent the chemical
fogging of the emdsion. Others have used stripping film,
mliich is provided with a thin base which is said to protect the
emulsion, Pelc ( '47), Boyd and Williams ( '48) and RfacDonald
( '49). Holt and Warren ( '50) overcame the problem by providing ail a i r g a p between the tissue and the emulsion. F u r thermore, Boyd ('49) reported that control tissues fixed in
Bouin 's solution and placed directly upon NTR emulsion
caused no chemical reduction.
' This investigation was supported iu part by a research grant (C1681) from The
Xational Cancer Institute of the National Institutes of Health, Public Health
Service.
25
26
NEWTON E. EVERETT A N D EABBABA S. SIILIMOPU’S
I n our experience certain of these methods have not been
effective in preventing chemical fogging hence it seemed advisable to report our preliminary observations relating to the
cause and prevention of histochemical reduction of photographic emulsion by r a t tissues. Our results do not support the
observations of Board (’51) that tissue sulfhydryl compounds
a r e primarily responsible for this chemical reduction.
The studies reported here include : (1) Observations on the
effectiveness of various fixatives ‘and protective plastic films in
preventing the tissue reduction of emulsion ; ( 2 ) a comparison
of different photographic emulsions in their sensitivity to
tissue reducing substances ; ( 3 ) attempts to ascertain the
molecular size of these substances; and (4)tests for the proposals that the tissue sulfhydryl compounds are responsible for
the reduction.
EXPERIMESTS
Small intestine, ovary, testis, hyaliiie cartilage, liver, kidney
and brain of Sprague Dawlep rats were fixed in Carnoy’s fluid,
in Bouin’s solution, or prepared by the freeze-dry method. The
tissues were from normal animals, and from rats receiving C14
or Fe50 labeled compounds. The Bouin’s fixed tissues were imliedded in paraffin by routine methods and the Carnoy fixed
and the frozen dehydrated tissues were imbedded directly in
paraffin. Sections mere made and mounted d r y on Eastman
medium lantern plates. The application of slight pressure to
the sections with the finger mas effective in causing them to
adhere to the emulsion. The paraffin mas removed by xylene
and the slides were stored at 5°C. in light-tight boxes containing a drying agent. In addition to the fixed preparations, fresh
smears of the above tissues and of blood were made directly
upon several emulsions. The smears were air dried f o r 30
minutes, fixed in methyl alcohol for 5 minutes and stored as
above. The emulsions used for the smears included Eastman
Kodak lantern and NTR plates and Ilford lantern and mechanical plates. After from 14 days to three months’ exposure
the slides were developed by routine methods, keeping all solutions a t 18-20°C., and then stained with liematoxylin or witli
H I S T O C H E M I C A L RE DUCT ION O F EMULSION
27
Wright’s blood stain. The sections were then mounted under
a coverslip. All of the control tissues with the exception of
hyaline cartilage produced some reduction of the emulsion.
The reduction of emulsion by the tissue sections was reproducible and the specificity of this reaction for component tissue
parts is illustrated by the reduction associated with the intestinal villi (fig. 1) and with a n ovarian follicle (fig. 2 ) . The
mononuclear lencocytes were the only blood cells whicli effected a clear cut reduction of the emulsion and this reduction
was variable (fig. 3 ) . I n some instances the lymphocytes produced a pronounced hlackeniiig of the film and in others little
or none. The pattern and depth of the reduced silver grains in
the histochernographs could not be reliably differentiated from
that known to be prodnced by beta particles.
It was observed that certain of the emulsions were much
more responsive to the histochemical reduction than others.
F o r example, the lymphocytes of control bloocl produced an
intense blackening of Tlford 1,anternplates, considerable reduction of the N T B plates and minimal reductioii of the Ilford
mechanical plates. The response of the Eastman Kodak lantern plates to histochemical reduction by blood cells has been
variable. Slides received and used prior to the last pear were
blackened by lymphocytes whereas the more recent shipments
of slides have shown little or no reduction by these cells. More
recent experiments indicate that this reduction by control blood
smears can be reduced by fixing the smears in methyl alcohol
after a minimal drying period of approximately 5 minutes.
I n view of the reduction associated with the control sections
attempts were made to protect the emulsion from reducing
substances within the tissue. Sections of the same tissues were
mounted d r y upon glass slides and the paraffin was removed b:xylene. The slides were dipped in absolute eythl alcohol ancl
then in 1%nitrocellulose in ether-alcohol. After drying f o r
24 hours, the sections were coated with either Dnco cement,2
E. I. du Pont de Nemours Co., Wilmington, Delaware.
28
NEWTON B. EVERETT AND BARBARA S. SIMlRIOSS
~ i l i c o n ,Geoii
~
R e ~ i i i ,Permamount
~
;t o r Formvar.6 Some of
the sections were covered with cellophane, which was 27 p iii
thickness. The slides were dried again for 24 hours and then
coated with Ansco Bulk Eniulsioii A. These were stored as
above for one to three months and developed by routine
methods. The sections were stained in a weak solution of heniatosylin and mounted as above. Examination revealed that with
the exception of the cellophane the various protective coatings
emplmoyed were ineffective in premnting the reduction of the
einulsion by the control tissues.
The period of two weeks was about the minimal exposure
time that produced a noticeable reduction of the emulsion. I n
general the longer the exposure period the greater was the
reduction.
I n view of previous satisfactory experience with Bouin's
fixed tissues mounted on emulsion by floating the sections in
water, it was felt that the water had probably been effective in
removing or inactivating the silver reducing substances. This
was confirined by subjecting sections of Bouin ' s , Carnoy 's,
formalin and alcohol fixed tissues to large volumes of water for
30 minutes before placing theiii in contact with the various
emulsions. After threcl months' exposure control tissues
mounted iii this may failed to show any noticeable reduction of
any emulsion used. Recent efforts to use control sections of
Carbowax imbedded tissues mounted d r y on emulsion have
shown that these produced a marked blackening of the emulsion within one week, however if before mounting 011 emulsion
the tissue sections were subjected for 30 minutes each to three
changes of water there was no perceptible reduction within 60
days.
Dri Filiii no. 987, General Electric Co., Selieiirctady, K. Y .
' Type 200 X 20, B. F. Goodrich Co., Cleveland, Ohio.
'A synthetic resin, Fisher Scientific Co., S e w Tork, $7.
Y.
A thermoplastic polyvinyl forni:il, Shawiiiipan Products Corporation, S e w York,
N. Y .
HISTOCHEMICAL.
R E D U C T I O N O F EMULSIOX
29
I n further attempts to characterize the molecular size distribution of the substances in normal tissues responsible f o r reduction of emulsion, multiple organs from a control rat were
fixed in Carnoy's fluid for three hours, passed through three
changes of absolmute alcohol and two of chloroform and dried
by evaporation. The dried organs were ground with a mortar
and pestle, and dialyzed 54 hours at 5°C. against several
changes of water in volumes 1000 times that of the sample.
Although the dialysate was not tested the non-dialyzable fraction retained its emulsion reducing capacity.
T o test for the water solubility of the reducing substances
ground Cariioy fixed tissues were extracted in warm water for
two hours and filtered. The residue was waslied thoroughly
with water in a suction filter, and was sprinkled on liquefied
emu1,sioiior on moistened film. The filtrate was lyophilized ancl
this fraction was also mountecl on photographic plates. After
one month exposure the lyophilized filtrate had produced a
considerable reduction of the emulsion, whereas the residue
caused no perceptible film blackeiiing.
Board's proposal ( '51) that the tissue sulflij-clrpl compounds
a r e responsible f o r the histocliemical reduction of photographie
emulsion was tested by utilizing the red sulfhydryl reagent,
1-(4-chloromei~curiphenylazo)-naplitliol-2, which binds specifically with sulfhydryl groups giving a pink to red color (Bennett, '48, '51). Dimethyl forniamide was used a s the solvent f o r
the reagent in the present stud>-. Tliis metliocl has been shown
to bind all of the sulfhydr>d groups in muscle (Bennett, unpublished d a t a ) . Sections of Cariioy fixed ancl frozen deli>-dratecl testis, liver, skin, intestine, adrenal and kidney were
treated with this reagent. Sections so treated were then dried,
and corcrecl with Ansco Bulk Emulsion A, anel developed after
30 days' exposure. The pattern of reduction produced by these
tissues was the same as that produced by the non treated tissues. Furthermore tlie sites of the strongest sulfliydryl reaction dicl not coincide with the areas of the tissue which produced the greatest reduction in the emulsion (fig. 4).
30
NEWTON B. EVERETT A N D BABBARA S. SIMMONS
DISCUSSION
The observations reported here emphasize some of the complications encountered in the radioautographic technique when
applied to tissues. It would appear that one cannot rely upon
certain of the conventional methods that have been used to prevent histochemical reduction of the emulsion. This is particularly true when l,ow concentrations of radioisotopes a r e used,
which necessitate long exposures to produce satisfactory radioautographs. Unfortunately the methods found to be most effective f o r protecting the emulsion against tissue reducing
substances, such as the use of stripping film o r interposed
sheets of cellophane, a r e not adaptable to localization of soft
beta emitters such a s C I 4 . Although the method of treating
tissues with large quantities of water prior to the contact of
tissue and emulsion appears to remove the reducing substances, this method is not applicable when the labeled tissue
compounds a r e water soluble.
The studies reported here show that tissues ~ 7 i l continue
l
to
reduce photographic emulsion although the tissue sulfhydryl
groups a r e bound by the red sulfhydryl reagent. Furthermore
the greatest reduction by the tissues so treated was from areas
where the sulfhydryl reaction was not pronounced. Although
some sulfhydryl, compounds reduce photographic emulsion
under certain conditions (Board, 51),it is questionable that
the sulfhydryl radical is entirely responsible for the reduction.
Other reducing radicals in tissues such a s the amine or phenol
groups a r e possibilities. Russel ('08) and Keenan ('26) observed that plant tissues would affect photographic plates
and concluded that the active substance was hydrogen peroxide.
The current observation that tlie reducing substances affect
photographic emulsion through interposed celloidin and other
films indicates a diffusion process. This is in line with the
observations of Drombromsky (cited by Keenan, '26) that
celluloid, gelatin and collodion did not protect photographic
film from the active substances in various metals.
HISTOCHEMICAL REDUCTION O F EMULSION
31
The report by Doniacli and Pelc ('50) a n d Bourne ('52)
that the reduction of emulsion by histochemical phenomena
and by nuclear phenomena can be reliably differentiated by
tlie pattern and depth of the reduced silver grains could not be
confirmed. The histochemical reduction described here occurs
in most instances only after prolonged exposure and would not
ordinarily be mistaken for radio-reduction if adequate controls
were used. However, since this type of reduction does place
certain liniitations upon use of the radioautographic technique
it is desirable to identify the substances responsible so if need
be they might be inactivated or removed from tlie tissues.
SUMMARY
A variety of normal rat tissues, fixed variously have been
shou~nto reduce photographic emulsions in a ~ 7 a yt h a t simulates reduction by beta particles. This reduction is not prevented during long exposui*esby coating the tissue sections o r
smears with nitrocellulose, I)uco cenient, silicon, a e o n Resin
o r Formvar. Tlie results iiidicate that these reducing substances diffuse from the tissues through the coating materials
into the pliotog~*apliicemulsion. Exposure of the tissue sections to large volunies of watei. prioi. to mounting was the most
effective method employed ill prevcntiiig this reduetion. Wa t e r
~
in the chemical resoluble reducing substances a i implicated
duction since the reduciiig properties were shown to reside in
lyophilized water extract of the tissue. Evidence is presented
that for the tissues used the sulfliydryl groups a r e not priniaiily respoiisihlc for the i.eductioii.
LTTERATUIIE CT'I'ED
T:FVXFTT,
1%. S. 19.51 The tleirronstratioii of thiol groups 111 certain tissues by
means of a i ~ colored
m
sulflipdiyl rrageiit. Anat. Rec., 110: 231-247.
Y,>.Nxm~r, 1%. S., A N D D. A . YPHANTIS 1918 1-( ~-Cliloroiiiercuriphenyl-A,O)
iiaplithol-2. J.A.C.S., SO: 3 3 2 2 .
I:o~RD, F. A . 1951 Sulfli;).dryl detection 1):- liistochciiiograpli~~.J. Cell. and
("oii~p.Pliysiol., 38: 3ii-387.
BOURNE,
G. 11. 1952 Autoradiograpliy. Biol. Rex., ? i :108-131.
ROYD,G . A,, ~ S DA. T. WILLIAMS 1948 Strip1,ing film teclinics f o r histological
:iiitoindiog.1.nl)lis. Proc. SOC. Riol. ancl ; \ I d . , 69 : 223-23'7.
32
NEWTON B. EVERETT A N D BARBARA S. SIMMONS
BOYD,G . A., AND F. A. BOARD 1949 A prelimiiiary report on histochemography.
Science, 210: 586-588.
DONIACH,I., AND S. R. PELC 1950 Autoradiograph technique. Brit. J. Radiol.,
5 3 : 184-192.
EIDINOFF,M. L., P. J. FITZGERALD,
E. B. f i I l I N E L A X D J. E. KNOLL 1951 I u t r a cellular localization of compounds labeled with tritium, IT by radioautography. Proc. Soc. Exp. Riol. and hfed., 7 7 : 225-229.
GROSS, J., R. BOGOROCH,
K. J. NADLERAND c. P. LEBLOND 1951 The theory and
niethods of the radioantographic localization of radioeleinents i n tissues.
Am. J. Roentgenol., 65 : 420-458.
HOLT, 31. W., AND S. WARREN 1950 A radioautographic method f o r detailed
localization of radioactive isotopes in tissues without isotope loss. Proc.
Soc. Exp. Riol. and bled., 7 5 : 545-519.
KEENAX,G. L. 1926 Substances which affect photographic plates in the dark.
Chem. Rev., 3 : 95-1 11.
JrACDONALD, A. M., J. CORE, A. I<. SOLOMON A X D D. STEINBERG 1949 Stripping
filni technic f o r ratlioautographs. Proc. Soc. Exp. Biol. and Med., 72:
117-121.
PELC,S. R. 1947 Autoradiograph technique. Kature, 1 6 0 : 749-750.
RUSSELL,W. J. 1908 The action of resin and allied bodies on a photographie
plate in the dark. Proe. liop. Soc. (Lontl.) B, 8 0 : 376-387.
WILLIAXS,A. I. 1951 Xethod for prevention of leaching aud foggiiig iii antoradiographs. ~ucleoiiics,8 : 10-14.
PLATE 1
E S P L A N l T I O N OF F I G C R E S
I Ilistochemograph of iiitestiuiil villi from IiewlJorn rat. Carnoy fixed, paraffin
imbedded, sections iuouuted dry on E. li. nredium lantern slide. Fourteen
days’ exposure. X 4.20.
2
1Iistochemograpli of ovariaii follicle from adult rat. Frozen-dried, paraffin
imbedded, sections rnouiited dry o n E. Ii. medium lantern slide. Fourteen
days’ exposure. S o t e th:it the reduced silver grains are confined t o the area
orerlying the follicular cells. X 440.
3
Histochernograph of monoiiuclenr leucocytes. Elooil smear made directly on
Ilford lantern slide. Six weeks’ exposure. x 970.
HISTOC'HEMIC.\L ItED E C T I O N OF EJIULSION
S P \ V T O N R. Ir.\ G R h T T A K D BAKRAKA S. 81XlAIOKS
33
I’LATE 2
ESPLAN.\TION
OF F I G U R E S
4 S e w b o r n rat skin, (‘ariioy fixed, tre:ited with red sulfliydryl reageiit a i i t l c.o:itc(l
with Aiisco Bulk emulsion A . l’no nioiitlis’ exposure. The coloring i i i tlic q i i dermis and in the deeply 1il:icr.d skeletal niiiscle fibers i s (liic t o l r o u i i d r c ~ l
sulfliydryl rc:igeiit. S o t e t h a t the most intense silrer reduction, iiitlic.:rtc(l 1)y
tlic stippling, is ;it t l i c ,jiiiic4ioii of the deriiiis and epidermis.
34
HISTO(’EIEMIC.~IJ RE1)UCTION O F EMLLSIOii
PL.ITF: 2
U K W T O S B. E V E R E T T A K D R A R B A R A S . S I M M O XS
35
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