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 FEW 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% 100 ml formalin..... 40 ml acetic acid (glacial).... 10 ml picric acid 2 g Picric-acetic-alcohol ethanol, 80% 100 ml acetic acid (glacial).... 10 ml picric acid 2 g 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 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 37 0 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. 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 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, 245- and 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 Acids i. Acetic Reagent 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 26. Sodium carbonate 27. Sodium hydroxide Various Concentration or composition Formic acid 50%. Na citrate 20%. Equal volumes of each Normal 2 to 10 drops HNO3 in SO ml water 0-5% 3-5% 1% Saturated Saturated 5 % w/v aqueous 100% 100% 1 % w/v aqueous Saturated 10% 1 minim per ml, aqueous 10% formalin neutralized with MgCOa 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 5% soln. 1% Conditions of test (room temperature unless contrary stated) Various 6 to ours 6 h or longer 18 h 37 0 C i8h37 C i8h37 C 1 h to several days 37 C 1 or 2 min Effect on stain Depresses reticular and nerve stain Suppresses costaining, especially of smooth muscle Increased reticularstain Affinity of nerve for stain increased Increased affinity of nerve-fibres for stain in sections fixed by formaldehyde Enhanced nerve stain in some cases Tends to loosen sections. Affinity of nerve for stain enhanced Slight tendency to loosen sections. May improve nerve stain slightly Sections lost from slides