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Use of anhydrous vapors in post-fixation and in staining of reactive groups of proteins in frozen-dried specimens for electron microscopic studies.

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Use of Anhydrous Vapors in Post-Fixation and in
Staining of Reactive Groups of Proteins in
Frozen-Dried Specimens for Electron
Microscopic Studies
ISIDORE GERSH, JUAN VERGARA AND GIOVANNI L. ROSS1
Department of Anatomy, University of Chicago, Chicago, Illinois, and
Catddra de Biologia, Universidad de Chile, Santiago, Chile
In 1953, Lamb and his colleagues described a method for reacting l-fluoro2,4-dinitrobenzene with cells. After some
6 successive steps, silver was introduced
into the molecule to enhance contrast for
electronmicrography. The Sanger reagent
was thought to react with free amino
groups, sulfhydryl groups, the phenolic
group of tyrosine, and the irnidazole group
of histidine in the cell proteins. Kendall
and Mercer ('58) developed a simpler
method for staining proteins in cells which
would be suitable for electron microscopy,
by the use of bromoacetate followed by
lead which is chelated with the reaction
product. Increased contrast caused by the
bound lead was regarded as indicating
sites of protein. Bahr and Moberger ('54)
and Bahr ('57) used solutions of methyl
mercuric chloride to enhance contrast of
sites rich in sulfhydryl groups. Apart from
questions of specificity, especially in the
first two methods, all seem to be of limited
potentiality in the sense that prolonged
immersion in the water of the reagent
solutions, and particularly the changes in
pH during the reactions, undoubtedly
disturb the submicroscopic structure.
In this paper, a general method is described for reacting tissue proteins (and
smaller units) in frozen-dried specimens
with certain anhydrous vapors which combine with reactive groups in the specimen.
The reactions all take place in vucuo. Enhanced contrast with these reagents is attributed to the increase in the mass which
they cause. A special variant of the general method is the reduction in vucuo of
organically bound copper in the vapor
phase in sites rich in catechol-amines like
adrenaline and noradrenaline. In this in-
stance the increased contrast in the electronmicrographs is attributed to copper.
Water is avoided in both the general and
the special methods in order to reduce diffusion, extraction, or dislocation of components of submicroscopic structures.
The general reagents, which are employed in the vapor phase in this study, are
the same as, or similar to, those used so
successfully by biochemists in their analyses of amino acid sequence in solutions
of proteins or polypeptides. They include :
l-fluor0-2,4-dinitrobenzene (DNFB), piodophenylsulfonyl chloride (PIPSYL) , 3,
4,5-triiodobenzoyl chloride (TIB), and
methyl mercuric chloride (MMC) . DNFB
is an oily liquid at room temperature,
while the others are solids. The use of
DNFB will be described in another report.
In vucuo, PIPSYL and MMC have an appreciable vapor pressure at 50-55°C or
below, while the vapor pressure of
TIB and CAA (copper acetyl acetonate) becomes appreciable at about 90°C. The
molecular weight of these substances is
significantly high: PIPSYL--302.5, TIB518.5, MMC-251. The molecular weight
of the corresponding reacted radicles is:
267, 483, and 215.5.
MATERIAL AND METHODS
Liver, adrenal medulla and epididymal
sperm of mice were used as test materials.
For the liver preparation, adult mice were
deprived of food and water for two days
prior to being killed by thoracotomy. The
liver was sliced about 0.1-0.2 mm thick
and frozen ultrarapidly in propane cooled
to about - 185°C in the manner described
by Gersh, Isenberg, Stephenson and Bondareff ('57). To obtain the adrenal medulla,
445
446
ISIDORE GERSH, JUAN VERGARA AND GIOVANNI L. ROSS1
normal adult male mice were anesthetized
by intraperitoneal injection of nembutal. The mice were then bled, and the
left adrenal gland was removed to a moist
box. The cortex was progressively sliced
away until it was thought very few cortical
cells remained. Then the gland was cut
into small pieces like the liver and frozen
in the same way. To obtain sperm smears,
adult mice were killed by bleeding, and
the epididymis was removed. After this
was slashed many times with a razor blade,
sperm smears were made on small squares
of thin aluminum foil and frozen ultrarapidly as described by Nelson ('58). All
specimens were stored in liquid nitrogen
in small glass vials stoppered with a cotton plug. For drying, the specimen vials
were transferred to tube C, S, or F, depending on the reagent, the lower part of each
being maintained at - 40°C or less by
means of a deep-freeze (see fig. 1 ) . After
drying, the specimens were exposed to
the reagents or vapors in the manner described below.
Tube C (for control) was used when the
specimen was to be heated and/or alcohol
treated only. Heat was applied by immersing the lower part in a steam or oil bath.
When the vacuum tube had cooled to room
temperature, 95% alcohol was admitted
through the thistle tube until the vials
were covered. The vacuum was broken
and the glass vial was removed and immersed in 95% alcohol in a Petri dish.
The specimens were loosened from the
foil and sliced into two or more parts.
Each was transferred to a glass-stoppered
micro-(Feigl) tube in 95% alcohol where
it remained for one day.
Tube S (for solid) was used to admit
vapors of PIPSYL, TIB, or CAA to the dry
specimen in vacuo. The lower part was
maintained at - 40°C or less, and the
specimen vial was removed from liquid
nitrogen and dropped into it. The reagent
was placed in the weighted reagent tube A
attached to the rotatable stop-cock B. The
plates were sealed with vacuum grease so
that the openings C and D were superimposed, and the whole was connected with
the vacuum line by turning stop-cock E.
The next day, the tube was allowed to reach
room temperature. Without breaking the
vacuum, stop-cock B was rotated so that
the weighted reagent tube slid down to the
level of the specimen vials. The tube was
then immersed in a steam or oil bath
(54°C for PIPSYL, about 95°C for TIB
and CAA). When crystals first appeared
on the cooler walls above the level of the
bath, the vacuum was closed (stop-cock
E ) for 5 hours. After this time, the reagent tube was raised by turning stopcock B in the opposite direction, and the
specimens were warmed to 60°C for
PIPSYL and 100°C for TIB and CAA; during this time, the connection with the vacuum line was resumed until the next day.
The tube was then allowed to come to
room temperature again, the vacuum was
closed by turning stop-cock E, and the
rubber connection to the vacuum line was
removed. Enough 95% alcohol was admitted through the stop-cock E to cover
the specimen vials. The vacuum was
broken, and the specimens were kept for
one day in 95% alcohol as described above.
For successive treatment of specimens
with formaldehyde vapor and PIPSYL or
TIB. The weighted reagent tube A of tube
S was first filled with paraformaldehyde
powder, and the specimen was dried at a
low temperature in the usual manner.
Tube S was warmed to room temperature
and the reagent tube was lowered to the
level of the specimen vials. This part of
tube S was then heated by a steam or oil
bath at 100°C for 5 hours in a closed
vacuum. After this time, the reagent tube
was raised and the connection of tube S
to the vacuum was resumed until the next
day. Tube S was then cooled to room
temperature. The upper plate was slid to
one side so that the openings, C and D,
were closed. The vacuum in the upper
part could then be broken without affecting the vacuum in the remainder of tube
S. The upper part of the assembly was
separated and the unevaporated paraformaldehyde removed and replaced by the
protein reagent. The upper part of the
assembly was replaced and a vacuum reestablished in it. The upper plate was slid
over the lower until their openings
matched. The reagent was lowered to the
level of the specimen vials by rotating
stop-cock B and the tube was heated to
54°C (for PIPSYL) or 95°C (for TIB).
Subsequent manipulations were the same
as those already described.
CYTOCHEMICAL VAPOR STAINS FOR EM
447
TUBE F
Fig. 1 Diagram of glass parts used for freeze-drying of the specimens, and for the subsequent manipulations. The upper parts of each tube are reduced approximately 1/3 (tube
C ) , 1/4 (tube S), and 1/5 (tube F). See text, pp. 446, 448.
448
ISIDORE GERSH, JUAN VERGARA AND GIOVANNI L. ROSS1
For successive treatment of specimens ml water. The precipitate was filtered with
with acetic anhydride and PfPSYL OT TIB. suction, washed with water and dried. To
After the specimens in tube S were dried recrystallize, the dried precipitate was
at - 40°C or lower, acetic anhydride was dissolved in a minimum volume of benintroduced into the weighted reagent tube zene. After filtering, petroleum ether was
A. After 5 hours exposure at room tem- added gradually, with stirring, and the
perature, excess vapors were removed by crystals were washed with petroleum ether
the vacuum system. The next day, the and dried.'
Other protein reagents were also tested
solid reagent was introduced in the usual
way, and after the customary exposure at in preliminary trials, but they seemed to
the same temperatures, the specimens be not as promising as the reagents above.
were covered with 95% alcohol. Both These include 2,3,5-triiodobenzoyl chloride.
manipulations effected successive expo- tribromacetaldehyde, and trichloracetyl
sures of dried specimens to anhydrous chloride.
After 24 hours in 95% alcohol, the
formaldehyde or acetic anhydride vapors
and PIPSYL or TIB vapors without break- specimens were embedded in celloidin and
then in methacrylate according to the foling the vacuum.
lowing
schedule :
Tube F (modified after Finck, '58) is
Absolute alcohol-three changes in one
simpler in some ways. The specimen vials
were dried in the usual way, and the tube hour.
Alcohol-ether (equal parts) - three
was brought to room temperature. Air,
changes
in one hour.
passed through a silica gel column, was
Thin nitrocellulose (7% )-three hours.
slowly allowed to reenter the tube, and
Thick nitrocellulose (15% )-overnight.
the phosphorus pentoxide boat was reHardening of celloidin-one day.
moved. The specimen vial was tied to a
Chloroform vapor-overnight.
glass rod together with another vial conSpecimen blocks transferred to absotaining the reagent. Both were replaced in
the vacuum tube and the vacuum was re- lute alcohol-one hour.
25% methacrylate mixture (20 methyl:
instituted. The times of exposure to the
vapor and temperatures were the same as 80 butyl) in alcohol-one hour.
50% methacrylate mixture in alcoholthose given above. The exposure time with
MMC was 6 hours at room temperature. one hour.
75% methacrylate mixture in alcoholThe vacuum was broken each time a reagent was used, and as in the earlier pro- one hour.
Methacrylate mixture-one hour withcedures, the final denaturation of the
specimen was in 95% alcohol. Tube F out catalyst, several hours with catalyst.
Embed in prepolymerized methacrylate
was used in the preliminary work with
55°C.
at
DNFB, PIPSYL and TIB, and throughout
Sections were cut with a Porter-Blum
with MMC.
The sources of the protein reagents are ultramicrotome as nearly as possible of
as follows: PIPSYL was supplied by K and the same uniform thickness, floated on
K Laboratories of Jamaica, New York, and water, and mounted on formvar. The liver
MMC by Metalsalts Corp., Hawthorne, sections were photographed at 40 kv, the
New Jersey. MMC was purified by steam adrenal medulla sections at 60 kv, with
distillation before use. TIB was prepared a Philips electron microscope with a rated
from 3,4,5-triiodobenzoic acid (Eastman resolution of 20A, with 35 mm, SpectroKodak) by the method of KIemme and scopic film 649-GH. They were printed on
Hunter ('40). CAA was prepared in the the same grade of contrast paper at a
foIlowing way: 10 g of redistilled acetyl magnification of X 5. Exposure and
acetone (2,4-pentanedione) (Eastman printing times were as nearly constant as
Kodak) was suspended in 40 ml water, possible. Sections of the sperm were photoand 6N ammonium hydroxide was added graphed with an Elmiskop I.
dropwise with stirring until solution was
'This reagent may also be purchased from
practically complete. To this was added MacKenzie Chemical Works, Inc., Central Islip,
a solution of 5 gm of cupric nitrate in 20 N. Y.
CYTOCHEMICAL VAPOR STAINS FOR EM
RESULTS
449
are treated with either of these compounds
and later with one of the vapor stains,
salient features are the same as those de- the ground substance appears appreciably
scribed earlier by Gersh, Isenberg, Bon- less dense than in the specimens treated
dareff and Stephenson ('57) : The ground only with the stain, though denser than in
substance of the cytoplasm consists of the control (untreated) specimens.
Comparison of sections of unstained
numerous submicroscopic vacuoles whose
walls are denser and continuous through- sperm tail with those stained in PQCUO
out. In small regions, frequently in rela- with MMC vapors shows enhanced contion to homogeneous-appearing mitochon- trast in the latter, especially in the longidria, are denser regions which are con- tudinal filaments and the helical coil, but
fined to the correspondingly thicker walls also in the matrix of the sperm tail and in
of submicroscopic vacuoles. These regions the residue of the intercellular substance
correspond to the ergastoplasm of osmium- (see figs. 6 and 7). These findings resemfixed liver, and have been shown to con- ble those which Nelson ('60) made with
tain numerous granules which may be other sulfhydryl reagents.
selectively stained with gallocyanine-chroIn the cells of the unstained adrenal
malum (Finck, '58). The vacuolar struc- medulla, the cytoplasm and the nucleus
ture of the nucleus resembles that of the consist of submicroscopic vacuoles and
cytoplasm, but the walls are thicker and their walls, as in the liver. Unlike the liver
denser in larger areas, corresponding to cells, the adrenal medullary cells contain
chromatin as seen with the light micro- numerous small and large granules of
scope. In some regions, the vacuolar walls low density (fig. 8). These are where the
are so thick as to appear to obliterate the greatest change in density takes place
vacuoles. The nucleolus appears to be after treatment with CAA vapors (fig. 9).
less dense than the chromatin and to This increased contrast is largely oblitercomprise numerous vacuoles. Both chro- ated when the cells are derived from mice
matin and nucleolus contain many gran- stimulated excessively by injections of
ules which may also be stained with gallo- insulin. The large granules correspond in
cyanine-chromalum (Finck, '58).
size with those separated centrifugally by
These differences in density largely van- Hillarp and his co-workers ('54).
ish after the specimens are stained in
DISCUSSION
UQCUO with anhydrous vapors of PIPSYL
There are several advantages in the use
or TIB. This is undoubtedly due to the
enhanced contrast of the protein between of protein reagents in the gaseous state as
the basophile granules of both the nucleus stains for electron microscopy, more parand the cytoplasm. This enhanced contrast ticularly when the specimens are in the
or increased density of the ground sub- dry state. By comparison with the use of
stance of the protoplasm may be clearly aqueous or alcoholic solutions of the
observed by comparing figure 2 (unstained) same reagents, there is bound to be less
with figure 3 (stained with TIB) and fig- diffusion, extraction, or displacement of
ures 4 and 5 (stained with PIPSYL). There the components of submicroscopic strucare thus two indicators that combination tures, or even of the structures themselves.
of these compounds with proteins has In addition, when the reagents combine
increased their density: (1) the decrease with the reactive groups of proteins, the
in relative prominence within a single reaction products are less soluble. For this
section of the denser regions rich in baso- reason, it is even possible that, in addition
philic material, and ( 2 ) the increased to proteins, small polypeptides may be
density of the ground substance of stained preserved. In addition, it is possible that
material in comparison with the density combination of these reagents with amine
of the unstained ground substance in groups of proteins may make the protein complex hydrophobic and reduce the
other sections.
Formaldehyde and acetic anhydride tend swelling tendency of proteins in aqueous
to block amino and other groups. It is solutions. Whether for these reasons or
not surprising, then, that when specimens for others, it is clear that aqueous gallo-
In the heat-and-alcohol-treated liver, the
450
ISIDORE GERSH, JUAN VERGARA AND GIOVANNI
cyanine-chromalum, alone or preceded by
ribonuclease or deoxyribonuclease, results
in very good preservation of submicroscopic structure in specimens pretreated
with the vapor reagents. This has been
observed in preliminary studies of liver,
and in more complete studies by Mundkur
of basophilia in yeast.
For lack of information, it is impossible
to state categorically at this time with
which reactive groups of proteins or polypeptides these reagents combine when they
are in the vapor phase and the protein in
the solid state. Information is, however,
available, on which reactive groups are
affected by the reagents when both are
in solution. The remainder of this paragraph is based mainly on the pertinent reviews of Stein and Moore ('46), Herriot
('47), Olcott and Fraenkel-Conrat ('47),
Sanger ('55), and Anfinsen and Redfield
('56). DNFB combines with terminal and
e amino groups (though not always completely) and with imidazole, sulfhydryl and
phenolic groups. Greater specificity may
be achieved with certain reagents by strict
control of the conditions of the reaction
(concentrations, temperature, time, pH,
ionic strength, etc.). But even so, this
specificity may not apply to other proteins.
In the absence of biochemical analysis, it
seems to be not possible to limit or define
the specific reactive groups of any particular protein which combines with DNFB; it
is possible only where biochemical analysis
is performed on purified proteins. This
degree of certainty is difficult or impossible
to achieve with most of the cell proteins
in situ in a cell. Similar uncertainties as
to specificity are common also to PIPSYL
and TIB. By contrast, MMC seems to be
quite specific for sulfhydryl groups, at
least under certain conditions, and its use
has the further advantage that it is small
(Bahr and Moberger, '54; Bahr, '57; and
Bennett and Watts, '58).
This brief summary should clarify the
probable general degree of specificity (or
lack of it) which is to be encountered under the conditions of our experiments,
where the reactions take place in vacuo in
an anhydrous solid-vapor phase system.
The two primary reagents (PIPSYL and
TIB) probably react with free amino
groups, but may react with others, while
L. ROSSI
MMC may be specific for sulfhydryl
groups.
In the copper acetyl acetonate vapor, intended for the detection of catechol
amines in the adrenal medulla, the ligand
bonds of the copper are broken and the
copper is reduced to the inorganic state, in
which state it is probably indiffusible and
not extractable by commonly used solvents. Strictly speaking, this reaction is
probably not specific for the catechol
amines, as other substances (such as ascorbic acid, sulfhydryl and unsaturated
fatty acids) also reduce the organically
bound copper. However, the reaction in
the cells of the adrenal medulla may be
regarded as virtually specific because of
the high concentration of the catechol
amines and the correspondingly high concentration of copper in specific submicroscopic structures, in comparison with the
slight and uniform enhancement of background contrast caused by all other reducing substances.
It has been stated by several researchers that, apart from any other considerations, the fine structure of material prepared by freezing and drying must be
distorted very greatly because of the supposed disturbance or gale caused by the
reentry of air into the vacuum system at
the end of a n experiment. This objection
has been refuted in the past by the fact
that there is no sign of such a violent disturbance after post-fixation in vacuo by
vapors of alcohol, osmium, iodine, etc., or
when the alcohol vapors are slowly condensed in the dehydration chamber before
the vacuum is broken. Now it is possible
to add also that the submicroscopic structures remain the same when they are
hardened in vacuo with vapors of PIPSYL
or TIB.
Because of the very useful post-fixation
and staining properties of these three reagents in their vapor phase, they seem
promising in certain problems where the
concept of state or degree of aggregation
is concerned. In preliminary studies of
developing rat tail tendon, for example,
where it is extremely difficult to preserve
and stain the ground substance of the
connective tissue, very well-fixed specimens, more deeply stained than was hitherto possible, have been observed. A
CYTOCHEMICAL VAPOR STAINS FOR EM
similar observation has also been made
by Molnar in the osteoid of developing
bone. The preservation of the submicroscopic structure in these instances is probably to be attributed to the insolubility of
the combination of the reagent (applied in
the gaseous state) with some of the water
and alcohol soluble components of the
ground substance. The enhanced stainability is probably to be attributed to the
probably larger number of free amino
groups available in such disaggregated
ground substance than in highly aggregated ground substance or in fully formed
collagen. This is comparable to the finding that in gelatin there are more terminal
amino groups than in collagen (Green,
Ang and Lam, '53).
Metals were used in earlier work to
stain frozen-dried specimens. Despite their
wide range over the periodic table, all of
those which resulted in enhanced contrast may be assumed to have produced
their effect by the same mechanism, i.e.,
primarily by combination with acid, imidazole, hydroxyl, sulfhydryl, and perhaps
other groups. The single basic feature
which the metal stains emphasize is
that ground substance of protoplasm
comprises numerous submicroscopic vacuoles of low contrast or density, separated from each other and enclosed by
denser walls. It should be emphasized that the contents of the submicroscopic vacuoles were only less dense
than their walls, but not empty. With
the general protein stains (PIPSYL and
TIB), whose use is described in this report,
the same basic feature of protoplasm appears, but the contrast between the contents of the vacuoles and their walls is
less marked. This is a consequence of the
fact that the proteins of the submicroscopic vacuoles are relatively more deeply
stained than are those of the walls, with
an apparent resultant loss in prominence
of the walls. These general protein reagents all react primarily with terminal
and c amino groups, and also perhaps
with others. One may deduce, then, that
more such reactive groups occur in the
contents of the vacuoles than in their
walls, due probably to the less dense (or
more disaggregated) state of the proteins
in the former than in the latter. Sjo-
451
strand and Baker ('58) have claimed that
the submicroscopic vacuoles as observed
in unstained or metal-stained preparations
are small ice crystals. The denser staining
of the contents of the submicroscopic
vacuoles achieved by the general protein
reagents disposes of this interpretation,
for if there were ice crystals, the spaces
occupied by them would remain unstained.
MMC in the gas phase combines with
sulfhydryl groups apparently as specifically as the colored compounds used by
Bennett (see Bennett and Watts, '58) and
by Barnett, Tsou and Seligman ('55). It
has the additional advantage that it is
smaller, and hence is less restricted by
steric hindrances. In conjunction with the
particular method employed, there is the
possibility that prior treatment of the
specimen in Z ~ U C U Owith B-mercaptoethanol
gas may be of use in indicating sites rich
in disulfide bonds.
As stated above, while CAA is not
specific for catechol amines, it is useful
in the adrenal medulla for the identification of sites where such compounds are
concentrated. These sites are preformed
granules which probably correspond with
those described and separated by Hillarp,
Hokefelt and Nilson ('54), and Hillarp
and Nilson ('54). The discrimination of
the granules by the use of CAA in the
vapor phase is clearly more differential
than that achieved by others with aqueous
solutions of osmium tetroxide (Sjostrand
and Wetzstein, '56; de Robertis and Vaz
Ferreira, '57). This is so partly because
the ground substance of the cytoplasm is
less dense in the frozen-dried material and
the reagent more specific, and partly because diffusion of the catechol amines is
avoided.
It is necessary to mention two general
features of importance in cytochemical
studies with the electron microscope. The
first is that, all other factors being kept
constant, the background density of unstained frozen-dried material is minimal
after freezing and drying, except in those
sites where substances are dissolved by
the use of post-fixatives. Against this
minimal background density, small differences in density or mass as a result of
cytochemical testing are more likely to be
detected with the electron microscope than
452
ISIDORE GERSH, J U A N VERGARA AND GIOVANNI L. ROSS1
when other hatives are used. The second
general feature concerns colored reaction
products in cytochemical tests. While
these are necessary for light microscopy
(with some exceptions), and advantageous
as a control in electron microscopy, they
are not essential. Providing the increase
in mass is appreciable, it is of no consequence for electron microscopy whether
the reaction product is colored or colorless.
Removal of this restriction on cytochemistry expands enormously the possible applications of cytochemical techniques.
SUMMARY
A general series of reactions is described
for the study with the electron microscope
of protein sites in frozen-dried material.
The reactions all take place in vacuo, are
anhydrous, and are of a solid-gas two
phase nature. The main reagents combine
with amine (and probably other) groups.
They are p-iodophenylsulfonyl chloride
(PIPSYL), and 3,4,5-triiodobenzoyl chloride (TIB). The distribution of the reactive groups of proteins (and probably polypeptides) in liver cells is described. A
third reagent, methyl mercuric chloride
(MMC), has some specificity for sulfhydry1 groups, and was studied in sperm
tail. A special solid-gas reaction is that of
copper acetyl acetonate. Copper is deposited from the vapor phase in granules
of the adrenal medulla which are rich in
catechol amines.
ACKNOWLEDGMENTS
The work was supported by grants from
the Commonwealth Fund and from the
Clara A. and Wallace C. Abbott Memorial
Research Fund of the University of Chicago. The senior author is especially indebted to the following colleagues, all of
the University of Chicago, for numerous
helpful discussions: Dr. Kenneth D. Kopple, Department of Chemistry, and Drs.
Elwood V. Jensen and Herbert I. Jacobson, both of the Ben May Laboratories
and the Department of Biochemistry.
The authors are indebted to Mrs. Faustina lManelis for her devoted technical
work.
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Bahr, G. F. 1957 Changes in liver cell elements during stimulated protein synthesis.
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Bahr, G. F., and G. Moberger 1954 Methylmercury-chloride as a specific reagent for protein-bound sulfhydryl groups. Electron stains
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with reference to the storage and release of the
sympathomimetic substances. Acta Physiol.
Scand., 31, Suppl. 113: 79-107.
Hillarp, N-A.,
B. Hokfelt and B. Nilson 1954
The cytology of the adrenal medullary cells
with special reference to the storage and secretion of the sympathomimetic amines. Acta
Anat., 21: 155-167.
Kendall, P. A., and E. H. Mercer 1958 A
technique for staining intracellular protein.
J. Roy. Micr. SOC.,78: 40-43.
Klemme, C. J., and J. H. Hunter 1940 Synthesis of iodohippuric acids II-2,3,5-and 3.4,
5-triiodohippuric acids. J. Org. Chem., 5: 508511.
Lamb, W. G. P., J. Stuart-Webb, L. G. Bell, R.
Bovey and J. F. Danielli 1953 Specifications
for electron microscopy. Exp. Cell Res., 4:
159-163.
Molnar, 2. Personal communication.
Mundkur, B. Personal communication.
Nelson, L. 1958 Cytochemical studies with the
electron microscope I. Adenosinetriphosphatase
CYTOCHEMICAL VAPOR STAINS FOR E M
in rat spermatozoa. Biochim. Biophys. Acta,
27: 634-641.
1960 Cytochemical studies with the
electron microscope. 111. Sulfhydryl groups of
rat spermatozoa. J. Ultrastruct. Res., i n press.
Olcott, H. S., and H. Fraenkel-Conrat 1947
Specific group reagents for proteins. Chem.
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453
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Abbreviations to plate
NUC, nucleus
CHR, chromatin
ER, ergastoplasmic densities
M, mitochondria
FIL filaments of sperm tail
HEL, helical coil of sperm tail
PLATE 1
EXPLANATION O F FIGURES
454
2
Electronmicrograph of part of control liver cell of starved mouse. Prepared by freezingdrying, heat and alcohol post-fixation, unstained. The nucleus (NUC) consists of numerous pale submicroscopic vacuoles whose walls are somewhat denser. Chromatin
(CHR) is markedly denser. Submicroscopic vacuoles of the cytoplasm and their walls
are similarly pale. Denser parts (ER), especially around mitochondria ( M ) , are rich in
RNA and i n suitably stained specimens contain numerous granules. Such regions are
probably equivalent to the granular part of the ergastoplasm as observed after fixation
and staining in aqueous solutions of osmium tetroxide. X 42,000.
3
Electronmicrograph of part of liver cell of starved mouse. Prepared by freezing-drying
followed by treatment in V ~ C U O with vapors of 3,4,5-triiodobenzoyl chloride (TIB ).
This combines with certain reactive groups of proteins and causes enhanced density.
The submicroscopic vacuoles of both nucleus (NUC) and cytoplasm are denser than i n
the unstained controls, the contents of the vacuoles being frequently as dense as their
walls. The mitochondria ( M ) are also denser than i n the controls. The difference i n
contrast between the chromatin (CHR) and the remainder of the nucleus, and between
the cytoplasmic regions rich in RNA (ER) and the rest of the cytoplasm is not as
marked as in the controls. X 42,000.
CYTOCHEMICAL VAPOR STAINS FOR EM
Isidore Gersh, Juan Vergaxa and Giovanni L. R o d
PLATE 1
455
PLATE 2
EXPLANATION O F FIGURES
4 and 5
456
Electroninicrographs of parts of liver cells of starved mouse. Prepared by freezing-drying followed by treatment in nucuo with vapors of p-iodophenylsulfonyl
chloride (PIPSYL). As in the specimens treated with TIB (fig. 3 ) , the major
differences from the unstained controls are: denser staining of submicroscopic
vacuoles and their walls in both nucleus and cytoplasm, relatively greater increase of density i n the vacuoles than in their walls, increased density of mitochondria ( M ) , and decrease in contrast between the chromatin (CHR) and RNArich regions i n the cytoplasm (ER) and the adjacent submicroscopic vacuoles.
x 42,000.
CYTOCHEMICAL VAPOR STAINS FOR EM
Isidore Gersh, Juan Vergara and Giovanni L. Rossi
PLATE 2
457
PLATE 3
EXPLANATION O F FIGURES
6
Electronmicrograph of part of control mouse sperm tail. Frozen-dried, heat and alcohol
post-fixation, unstained. Compare with figure 7. x 20,000.
7
Electronmicrograph of part of mouse sperm tail. Prepared by freezing-drying, followed
by treatment in vucuo with vapors of methyl mercuric chloride (MMC). This combines
with sulfhydryl groups and causes enhanced contrast. A s compared with the control
specimens in figure 6 , the filaments (FIL) and the helical coil (HEL) are denser.
There is some increased density also in the matrix of the sperm tail and in the residue
of the extracellular epididymal fluid. x 20,000.
8
Electronmicrograph of part of cytoplasm of adrenal medulla cell. Frozen-dried, heat and
alcohol post-fixation, unstained. The cytoplasm contains numerous submicroscopic
granules of low density. x 26,000.
9 Electronmicrograph of part of cytoplasm of adrenal medulla cell. Prepared by freezingdrying, followed by treatment in ZJCZCUOwith vapors of copper acetyl acetonate (CAA).
Copper is deposited i n regions rich i n catechol amines (which include adrenaline and
noradrenaline), and causes increased contrast. There are numerous small, paler granules, and some larger dense ones. The dense granules seem to be aggregates of smaller
ones.
458
CYTOCHEMICAL VAPOR STAINS FOR EM
Isidore Gersh, Juan Vergara and Giovanni L. Rossi
PLATE 3
459
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