155
The staining of nervous elements by the Bodian method
I. The influence of factors preceding impregnation
By TERRY WILLIAMS
(From the Department of Anatomy, University College, Cardiff. Present address:
Department of Anatomy, The University, Manchester 13)
Summary
The stainability of neural elements is determined during the various stages of tissue
preparation which precede impregnation.
The hypothesis (Romanes, 1950) that variations in the physiological state of the
neurones at the time of fixation might influence the stain was examined. Feeding and
anaesthesia did not influence the staining of gastric nerve fibres under the conditions
of the experiments.
A study was made of fixing fluids for Bodian staining. A picric-acetic-alcohol
mixture and alcoholic Bouin were convenient and effective, each being superior to
formaldehyde-acetic-alcohol. For overnight fixation formaldehyde solution neutralized with calcium carbonate was satisfactory, but where prolonged fixation is permissible formaldehyde neutralized with excess of magnesium carbonate gave superior
results.
Attention was drawn to the probability that the final stain could be improved by
'pretreatment' of mounted sections before silvering. Dewaxed sections were treated
individually with 27 substances from a variety of chemical groups to determine the
effects on subsequent Bodian staining. Nitric and sulphuric acids, formaldehyde
solution neutralized with magnesium carbonate, methyl alcohol, and perhaps sodium
carbonate contribute in different ways towards better preparations. In particular
pretreatment with nitric acid suppresses the background stain, especially in smooth
muscle; sulphuric acid increases the number of fine nerve fibres displayed, and methyl
alcohol facilitates the staining (in formaldehyde-fixed tissues) of nerve fibres that are
otherwise difficult to demonstrate, such as those in the renal cortex, which are almost
certainly post-ganglionic sympathetic fibres.
Introduction
F E W attempts have been made to improve results with the Bodian technique by investigating the stages of tissue preparation which precede impregnation. Yet the stainability of the nervous elements is decided, as is
well known, before incubation.
For convenience this report will be divided into a study of :
(a) factors operating up to the stage of attaching the sections to slides; and
(b) treatment of the sections before they are placed in the proteinate
solution: this will be referred to as 'pretreatment'.
The physiological state of the nervous tissues
Romanes (1950) suggested that variations in the physiological state of
neurones at the time of fixation could account for some, at least, of the vagaries
[Quarterly Journal of Microscopical Science, Vol. 103, part 2, pp. 155-62, June 1962.]
156
Williams—Bodian method for nervous elements
seen in silver staining. I have failed to influence the silver staining of axons
in the stomach wall by varying the period which elapsed between feeding and
death (rat), by subjecting dogs to nembutal anaesthesia during 6 h, or by
using various anaesthetics to kill the animals (rat and guinea-pig).
Fixation
After using a great number of fixing agents, Bodian (1937) concluded that
'the most generally satisfactory' for his method was formaldehyde-aceticalcohol. Polley (1956) reopened the subject with studies of preparations made
with sections from random blocks in the laboratory. He found Lawrentjew's
and Bouin's fluids successful for peripheral tissues, and 10% formaldehydesaline with excess calcium carbonate 'generally successful' for the central
nervous system.
As a convenient fixative, facilitating Bodian stains for viscera (gut, spleen,
pancreas, and bladder in man and laboratory animals), I have found the
following solutions superior to formaldehyde-acetic-alcohol for the purpose:
Alcoholic Bouin
ethanol, 80%
formalin
.
.
acetic acid (glacial)
picric acid
Picric-acetic-alcohol
ethanol, 80%
acetic acid (glacial)
picric acid
.
.
.
.
.
.
.
.
.
.
.
100 ml
40 ml
10 ml
2g
100 ml
10 ml
2g
Where formaldehyde is used in the fixing fluid care should be taken to
preserve it free from acids which form in it as breakdown products: for this
purpose the formalin used has been 'neutralized' by calcium or magnesium
carbonate (Stowell, 1941; Holmes, 1943). Where formalin alone is to be
employed as a fixative, formalin neutralized with calcium carbonate is to be
preferred for overnight fixation and with magnesium carbonate for prolonged
fixation. In the tissues the apparently limitless number of possible addition
reactions, plus the probability of chain reactions, makes it difficult even to
speculate on the chemistry of fixation by formaldehyde. Gough (1931), using
acetaldehyde as the fixative before a silver impregnation for glia, showed that
formaldehyde is not essential for silver staining. For the Bodian stain it is not
indispensable.
In the picric-acetic-alcohol mixture recommended, alcohol is the most
controvertible constituent: while it accelerates fixation, it may lead to patchy
fixation and staining. Even when it is practicable to perfuse through the blood
vessels, as Bodian (1937) recommended, the alcoholic fixatives often give
Williams—Bodian method for nervous elements
157
uneven staining results. Acetic acid may help the axon stain in accordance
with its tendency to encourage linkages between proteins and reagents,
although its value in fixing tissues before Bodian's method has been thought
to lie in its power to depress the argyrophilia of connective tissue. I am of the
opinion that picric acid increases the number of fine axons staining, particularly in smooth muscle, since these fine nerve-fibres stain badly when the
amount of picric acid is reduced.
When prolonged fixation is permissible, the greatest profusion of axons may
be displayed with Bodian's method by using formalin made alkaline by
standing over excess magnesium carbonate. Staining results after adding
excess of magnesium carbonate to the fixative were better than after all the
other additives tried, which included pyridine and a variety of alkalis. The
best results with mammalian stomach were achieved by the following method:
1. Dilute commercial formalin with 9 times its volume of tap-water.
2. Shake the solution with magnesium carbonate and allow to stand over an
excess of the salt.
3. Shake the fixative and do not allow the sediment to settle completely before
adding to the tissue. As a result a thin white coating of magnesium
carbonate lies on the tissue.
4. Fix for at least 3 weeks.
5. Dehydrate and embed by the usual procedure.
Pretreatment
Few records exist of attempts to improve the stainability of paraffin sections
by chemical treatment between attachment of sections to slides and impregnation. Aldehyde blocking agents have been used on account of the slender
evidence that aldehydes might affect silver staining (Lhotka and Davenport,
1950), and these substances have also been tested in the present investigation.
Some acids were also tried because there are precedents for the use of various
acids before commercial dyeing procedures and histological staining. Beyond
this the choice of pretreatments was governed by practical considerations and
guesswork. The former placed a limit on the number of compounds that
could be tested, and the guesswork was based on the hope that compounds
known to act on the nervous system, or which had proved useful as adjuncts
to other silver stains, might improve our staining results.
The following is a description of various chemical treatments applied to
paraffin sections before staining them by the usual Bodian procedure. Both
sections fixed with formaldehyde and with alcoholic Bouin were used in these
trials and, except where the contrary is stated, the effect of exposure of the
attached sections to a fluid for 12 h was observed. Sections were placed in
each fluid after dewaxing and passing down to water. After this pretreatment
the slides were impregnated separately. The chief results are recorded in
tabular form in the Appendix (p. 162).
158
Williams—Bodian method for nervous elements
Acids
Ehrlich added acetic acid to the dahlia used for staining mast cells, and
phenol is used in the Ziehl-Neelsen stain for tubercle bacilli. Nitric acid has
been used to increase the affinity of tissues for basic stains, but Mann (1902)
has found more intense staining after sulphuric acid.
Palmgren (1955) has achieved a nerve stain in the presence of smooth muscle,
or when argyrophil connective tissue is present, by using dilute nitric acid as
a pretreatment for the sections. The effects on the subsequent Bodian stain
of formic acid, periodic acid, acetic acid, nitric acid, hydrochloric acid, and
sulphuric acid have been tested, the last 4 in varying concentration.
Formic acid was used in the form of Bensley's decalcifier (equal volumes of
50% formic acid and 20% sodium citrate), which Romanes (1950) has found
useful in preparing sections for his own stain. There was no consistent improvement in selectivity afterwards.
It is perhaps surprising that Bensley's decalcifier did not accentuate
the reticular stain since it has been suggested that formic acid present in unneutralized formalin was responsible for the heavy reticular pattern seen
after the latter fixative.
Acetic acid. Holmes (1942) used a mixture of xylene and acetic acid as a
pretreatment for his silver method, in order to reduce the connective tissue
stain and to demyelinate axons which would not otherwise stain. He used
70% glacial acetic acid in xylene at 37° C for 48 h for this purpose. Bodian
(1936) on the contrary had recommended treatment of sections with only 5%
acetic acid overnight at room temperature. The great disparity between these
two recommendations is perplexing.
My experience, with acetic acid at concentrations of 5% and 70% and at
both room temperature and 370 C for periods up to 7 days, was that pretreatment with acetic acid makes no significant difference to the general pattern of
the Bodian stain. When sections are treated for days in 70% acetic acid, all
structures, nervous and non-nervous, appear paler in the subsequent stain.
Nitric acid. Davenport (1929) stated that Sand's (1908) contribution to
silver staining was to show that nitric acid suppressed the staining of cells and
particularly connective-tissue elements. He used a fixative which consisted
of 10% concentrated nitric acid in pure acetone and stated that the fairly
concentrated nitric acid mixture was the only fixative, except mercuric
chloride solution, which inhibited the staining of glia and other connectivetissue fibres. Ramon y Cajal (1933, p. 694) also used nitric acid in a fixative
with chloral and alcohol, referring to this mixture as 'Castro's decalcifying
fixer' in which 'the nitric acid, far from disturbing, seems to better the neurofibrillar action'.
It might be supposed that nitric acid could bring about the same improvements in a Bodian stain. Pretreatment with nitric acid suppresses the staining
of non-nervous elements, especially smooth muscle, connective tissues, and
red blood-corpuscles. These effects of nitric acid may be related to its
Williams—Bodian method for nervous elements
159
bleaching propensity; thus it is known to bleach formalin haematin pigment,
which is formed when formaldehyde acts on blood. We do not know why it
should suppress the staining of smooth muscle under conditions which do not
apparently impair the staining of nerves.
Above a critical concentration nitric acid interferes with nerve staining.
10 drops of concentrated nitric acid in 50 ml of water gives optimal results
when used for about 12 h.
Hydrochloric acid. The effect of pretreatment with dilute hydrochloric acid
was tried without any apparent effect.
Sulphuric acid. During immersion in dilute sulphuric acid bubbles of gas
form on sections. An increased affinity for nerve-fibres often follows. The
optimal concentration of sulphuric acid is 3 % to 5%. Prolonging the treatment from 12 h to several days does not spoil the subsequent stain.
Periodic acid. Pretreatment with 1 % periodic acid caused increased argyrophilia of connective tissue. This was not entirely unexpected, since Lhotka
(1953a) found that this reagent, used before silvering, brought about a
marked increase in the reticular argyrophilia obtained by Foot's ammoniated
silver carbonate technique (Foot, 1924, 1938; Foot and Menard, 1927). Further, Gridley (1951) used periodic oxidation in a silver method for reticulum,
based on the Bielschowsky technique.
Aldehyde blocking agents
Since periodic acid, an oxidizing agent with the ability to attack 1,2-glycol
linkages to produce aldehyde radicles, appears to increase the response of
reticular tissue to silver (see above), it might be thought that aldehyde radicles
normally present in smooth muscle-cells or connective tissue might be factors
impairing the Bodian stain. Lhotka and Davenport (1950) and Lhotka (19536)
suggested that aldehydes might play an important role in argyrophil phenomena. Lhotka (1954) then claimed that if tissue aldehydes are blocked before
silvering in the protargol technique, neurofibrillar argyrophilia is not appreciably affected. Later the same author (Lhotka, 1956) decided that aldehyde blocking reactions did, however, diminish reticular staining. It seemed
possible, therefore, that aldehyde blocking agents might be used to suppress
the reticular stain in sections of viscera, while the nerve-fibre stain would
persist. To test this, hydroxylamine HC1, 2,4-dinitrophenyl-carbazone,
and 2,4-dinitrophenylhydrazine were used separately in the pretreatment of
dewaxed sections, which were subsequently silvered according to the Bodian
method. Each reagent was used on two types of section, one being rat stomach
sections fixed in ordinary formalin and thus possessing a strong tendency
towards reticular staining, and the other dog stomach sections fixed in alcoholic Bouin made up with neutralized formalin and having little tendency
towards reticular staining. Hydroxylamine HC1 was used in a 1 % solution;
the others, being less soluble, were saturated. Sections were incubated in
these solutions at 37° for 18 h, washed in running water, and then stained.
160
Williams—Bodian method for nervous elements
Two control pairs of sections were included, one pair being incubated for
18 h in isotonic saline before staining and the other receiving no pretreatment.
No significant change in staining distribution was seen after pretreatment
with aldehyde blocking agents. None of these substances affected the intensity
of the reticular argyrophilic response any more than did isotonic saline under
the same conditions. This finding conflicts with that of Lhotka (1956). In
fact, sections soaked in isotonic saline were indistinguishable from those
treated for the same length of time in the aldehyde blocking solutions, but all
the soaked sections showed a diminished staining intensity when compared
with the sections which had been impregnated immediately after dewaxing
and taking down to water.
Miscellaneous
A number of other substances were tested for their capacity to affect the
subsequent staining reaction. Some of them are drugs known to act upon the
nervous system, others are reagents sometimes used in fixation in order to
facilitate nerve staining.
The results of these pretreatments are summarized in the Appendix and
need not be mentioned here.
Discussion
A few of the pretreatments yielded useful results. These were nitric acid
and sulphuric acids, formalin neutralized with magnesium carbonate, methanol, and perhaps sodium carbonate as well. These reagents contribute in
different ways towards a better result. Thus nitric acid appears to reduce the
staining of non-nervous elements, while sulphuric acid seems to increase the
number of fine nerve-fibres taking up the stain, particularly in smooth muscle.
Neutralized formalin can make good the deficiency in sections which had
initially been inadequately fixed in formalin. Pretreatment with methanol
is useful in providing a sharp nerve-fibre stain in some refractory tissues:
for instance it facilitates the display of nerve-fibres in the renal cortex,
which are almost certainly post-ganglionic sympathetic fibres and usually
resistant to the Bodian technique.
These findings strongly suggest that much can be done to improve the
stainability of valuable sections which have been inadequately fixed. Further
work may disclose better fixatives for the Bodian method, as well as pretreatments for displaying selectively different kinds of nerve-fibres.
References
BODIAN, D., 1936. 'A new method of staining nerve fibres and nerve endings in mounted
paraffin sections'. Anat. Rec, 65, 89.
1937. 'The staining of paraffin sections with activated protargol. The role of fixatives.
Ibid., 69, 153.
DAVENPORT, H. A., 1929. 'Silver impregnation of nerve fibres in celloidin sections.' Ibid.,
44, 79-
Williams—Bodian method for nervous elements
161
FOOT, N. C , 1924. 'A technic for demonstrating reticulum fibers in Zenker-fixed paraffin
sections.' J. Lab. clin. Med, 9, 777.
1938. 'Useful methods for routine examination of brain tumours.' Amer. J. Path., 14,
245and MENARD, M. D., 1927. 'A rapid method for the silver impregnation of reticulum.'
Arch. Path. Lab. Med., 4, 211.
GOUGH, J , 1931. 'Acetaldehyde as afixativefor neuroglia.' J. Path. Bact, 34, 419.
GRIDLEY, M. F., 1951. 'A modification of the silver impregnation method of staining reticular
fibers.' Amer. J. clin. Path., 21, 897.
HOLMES, W., 1942. 'A new method for the impregnation of nerve axons in mounted paraffin
sections.' J. Path. Bact., 54, 132.
1943. 'Silver staining of nerve axons in paraffin sections.' Anat. Rec, 86, 157.
LHOTKA, J. F., 1953a. 'Effects of oxidation on neurofibrillar argyrophilia.' Stain Tech.,
28, 101.
19536. 'Periodic-acid-Foot stain for connective tissue.' Ibid., 28, 129.
1954. 'The role of the aldehyde radical in neurofibrillar argyrophilia.' Ibid., 29, 293.
1956. 'On tissue argyrophilia.' Ibid., 31, 185.
and DAVENPORT, H. A., 1950. 'Staining similarity of Foot's and HIO 4 - Schiff technics.
Ibid., 25, 129.
MANN, G., 1902. Physiological histology. Oxford (Clarendon Press).
PALMGREN, A., 1955. 'Staining nerve fibres after sublimate-acetic and after Bouin's fluid.'
Stain Tech., 30, 31.
POIXEY, E. A., 1956. 'Silver staining of nerve tissue with a new silver proteinate.' Anat. Rec.
125, 509.
RAM6N Y CAJAL, S., 1933. Histology. London (Bailliere, Tindall, and Cox).
ROMANES, G. J., 1950. 'The staining of nerve fibres in paraffin sections with silver.' J. Anat.,
84, 104.
SAND, R., 1908. Arb. a. d. Neurol. Inst. a. d. Wiener Univ., 14, 339. Quoted by Davenport
(1929).
STOWELL, R. E., 1941. 'Effect on tissue volume of various methods of fixation, dehydration,
and embedding.' Stain Tech., 16, 67.
162
Williams—Bodian method for nervous elements
Appendix
Pretreatments and their observed effects
Concentration or
composition
Reagent
Acids
i. Acetic
2. Bensley's decalcifier
3. Hydrochloric
4. Nitric
5. Periodic
6. Sulphuric
Aldehyde blocking agents
7. Hydroxylamine HC1
8. 2-4 dinitrophenylcarbazone
9. 2-4 dinitrophenylhydrazine
Miscellaneous
10. Chloral hydrate
11. Methanol
12. Paraldehyde
13. Pot. bromide
14. Seconal (quinal barbitone)
15. Sodium barbitone
16. Tinct. opii
17. Formaldehyde
18. Pyridine
19. Acetic anhydride
20. Aniline oil
21. Caseinogen
22. Iodine
23. Methyl salicylate
24. Urea
25. Ammonia
Conditions of test
(room temperature
unless contrary
stated)
Effect on stain
Various
Various
Depresses reticular
and nerve stain
Formic acid 50%. Na citrate
20%. Equal volumes of
each
Normal
2 to 10 drops HNO3 in
SO ml water
12 h
Nil
12 h
6 to 12 hours
Nil
Suppresses costaining,
especially of smooth
muscle
Increased reticularstain
Affinity of nerve for
stain increased
0-5%
3-5%
12 h
6 h or longer
1%
Saturated
18 h 370 C
i8h37°C
Nil
Nil
Saturated
i8h37°C
Nil
5 % w/v aqueous
100%
12 h
12 h
100%
1 % w/v aqueous
Saturated
12 h
12 h
12 h
10%
Nil
12 h
Nil
12 h
1 h to several Enhanced nerve stain
days
in some cases
Nil
12 h
12 h
Nil
Nil
12 h
1 minim per ml, aqueous
10% formalin neutralized
with MgCO a
100%
100%
3 drops per 100 ml of 50%
ethanol
Excess in water
Saturated solutions in 95%
ethanol and 70% ethanol
followed by thiosulphate
solution
15 % w/v in ethanol
Saturated soln.
1 drop cone, ammonia in
100 ml water
12 h 3 7 ° C
12 h
12 h
12 h
1 or 2 min
26. Sodium carbonate
5% soln.
12 h
27. Sodium hydroxide
1%
12 h
Nil
Increased affinity of
nerve-fibres for stain
in sections fixed by
formaldehyde
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Tends to loosen sections. Affinity
of
nerve for stain enhanced
Slight tendency to
loosen sections. May
improve nerve stain
slightly
Sections lost from
slides