Selective Silver Impregnation of Degenerating Axons In The Central Nervous System

Rat and rabbit brains containing surgical lesions of 5-10 days' duration were fixed in 10% formalin (neutralized with calcium carbonate) for 1 week to 6 months. Frozen sections (15-20 n) were rinsed and then soaked 7 minutes in a 1.7% solution of strong ammonia in distilled water. Subsequent treatment was as follows: rinse; 0.05% aqueous potassium permanganate 5-15 minutes; 0.5% aqueous potassium metabisulfite, 2 changes of 2.5 minutes each; wash thoroughly in 3 changes distilled water; 1.5% aqueous silver nitrate, 0.5-1.0 hr.; 1% citric acid, 5-10 sec.; 2 changes distilled water; 1% sodium thiosulfate, 30 see.; 3 changes distilled water. Each section is then processed separately. Ammoniacal silver solution (450 mg. silver nitrate in 10 ml. distilled water; add 5 ml. ethanol; let cool to room temperature; add 1 ml. strong ammonia water and 0.9 ml. of 2.5% aqueous sodium hydroxide), 0.5-1.0 min. with gentle agitation. Reduction of about 1 minute is accomplished in: distilled water, 45 ml.; ethanol, 5 ml.; 10% formalin, 1.5 ml.; 1% citric acid, 1.5 ml. Rinsing; 1% sodium thiosulfate, 10 sec.; thorough washing followed by dehydration through graded alcohol and 3 changes of xylene or toluene complete the staining process. Normal nerve fibers are slightly stained to unstained, degenerating fibers, black. The treatment in potassium permanganate is critical since too little favors overstaining of normal fibers and too much abolishes staining of degenerating fibers.     

Silver Impregnation of Degenerating Axons in the Central Nervous System: A Modified Technic

Frozen sections of formalin-fixed brains containing surgical lesions, were treated with 15% ethanol for 0.5 hr., soaked in 0.5% phosphomolybdic acid for 0.25-1.0 hr., and subsequently treated with 0.05% potassium permanganate for 4-10 min. (The duration of the latter treatment is critical and individually variable). Subsequent procedure is as follows: decolorize in a mixture of equal parts of 1% hydroquinone and 1% oxalic acid; wash thoroughly and soak sections in 1.5% silver nitrate for 20-30 min.; ammoniacal silver nitrate (silver nitrate 0.9 g., distilled water 20 ml., pure ethanol 10 ml., strong ammonia 1.8 ml., 2.5% sodium hydroxide 1.5 ml.) 0.5-1.0 min.; reduce in acidified formalin (distilled water 400 ml., pure ethanol 45 ml., 1% citric acid 13.5 ml., 10% formalin 13.5 ml.) 1 min.; wash, and pass section through 1 % sodium thiosulf ate (0.5-1.0 min.); wash thoroughly and pass sections through graded alcohols and xylene (3 changes); cover in neutral synthetic resin.     

A Glycine-Hcl-Formalin Fixative for Improved Staining of Neuroglia

The pH of fixatives as well as the components of the fixative mixture exert a displacing action on the isoelectric point of tissue proteins, which influences the intensity of staining. Studies of these effects showed that ammonium nitrate, sulfate or chloride could be substituted successfully for ammonium bromide in Cajal's formalin-ammonium bromide fixative. However, the best results from staining with Rio Hortega's silver carbonate and with Cajal's gold-sublimate methods were obtained after fixation in a mixture consisting of: glycine, 1.05 gm; 12V HCl, 14.8 ml; concentrated formalin, 15 ml and distilled water to make 100 ml.     

Staining Pericellular Boutons of Glutaraldehyde-Fixed Nervous Tissue; A Chromation-Silvering Sequence for Frozen Sections

After fixing in phosphate-buffered 5% glutaraldehyde, pH 6.8, by perfusion, brains were sliced to 3-5 mm pieces which were placed in the fixative for 5-7 days. The pieces were washed through several changes of 2.26% NaH₂PO₄ for 12 hr, 30 μ frozen sections cut, and mordanted 2 days in an equal-parts mixture of 3.5% CrO₃ and 5% Na-tartrate, which had been aged at 20-25 C for 20 days prior to use. After washing in distilled water, the sections were put into a solution containing AgNO₃, 20 gm; and KNO₃, 15 gm, in distilled water, 80 ml; at 30 C for 1.5-2 hr, then reduced at 40-45 C in three pyrogallol solutions as follows: 1-2 sec in 1% pyrogallol in 55% alcohol; 3-4 sec in a 0.67% solution in 33% alcohol, and 5-7 sec in a 0.5% solution in 25% alcohol. Gold toning is optional; dehydration, clearing and covering, routine. The technic shows particularly the perisomatic fibers, boutons en passant and boutons termineaux. Fibers in nerve tracts may be visible but lightly stained; cell nuclei may be dark, but the cytoplasm remains pale.     

Basic Dye Counterstaining of Sections Impregnated by the Golgi-Cox Method

Celloidin blocks of Golgi-Cox impregnated material are cut at 50 μ, the sections collected in 70% alcohol, transferred to a 3:1 mixture of absolute alcohol and chloroform for 2 min, and then stored in xylene or toluene for at least 3 min, or up to 2 wk until processed further. Mounting is done on glass slides which have been coated with fresh egg albumen diluted in 0.2% ammonia water (or a 0.5% solution of dry powdered egg albumen) and then dried at 60°C overnight. For attachment to these coated slides, sections are first soaked for 2-3 min in a freshly prepared mixture of methyl benzoate, 50 ml; benzyl alcohol, 200 ml; chloroform, 150 ml; and then transferred quickly to the slides by means of a brush. After 2-3 min the chloroform evaporates and the celloidin softens. The slides are then immersed in toluene which hardens the celloidin and anchors the sections to the slides. Alcohols of descending concentrations to 40% are followed by alkalinizations, first in: absolute alcohol, 40 ml; strong ammonia water 60 ml, for 2 min, then in: absolute alcohol, 70 ml; strong ammonia water, 30 ml, for 1 hr. Excess alkali is then removed by 70% and 40% alcohol, 2 min each, and a 10 min wash in running tap water. Bleaching in 1% Na₂S₂O₃, for 10 min and washing again in tap water for 10 min completes the process preliminary to staining. The preparations are then stained for 90 min in an aqueous solution of either 0.5% cresylecht violet, neutral red, or Darrow red, buffered at pH 3.6. Dehydration and differentiation in ascending grades of alcohol, clearing with toluene or xylene, and applying a cover glass with a mounting medium having a refractive index of about 1.61 completes the process.     

Enhanced Reliability in Silver Impregnation of Terminal Axonal Degeneration-Original Nauta Method

Brains of rat with surgical lesions 3-5 days old are fixed in 10% neutralized formalin (excess of CaCO₃), 20 μ serial frozen sections cut therefrom and kept in neutralized formalin for an additional 24-48 hr. The sections are soaked in distilled water 12-24 hr, transferred to 50% alcohol containing 0.75 ml of concentrated NH₄OH (sp. gr. 0.91) per 100 ml 12-24 hr, placed in distilled water 2-3 hr and then in silver-pyridine solution (AgNO₃ 3% aq., 20 ml; pyridine, 1 ml) for 48 hr. Test sections are transferred directly to each one of 3 ammoniated silver-solutions, pH 12.8, 13.0 and 13.2, made as follows: To 200 ml of solution 1 (silver nitrate, 6.4 gm; alcohol 96%, 220 ml; NH₄OH (sp. gr. 0.91), 28 ml and distilled water, 440 ml) is added respectively 8-12 ml, 12-16 ml and 16-20 ml of solution 2 (2% NaOH) to give the pH desired. The test sections are studied and the optimal ammoniated silver solution chosen. Two baths of ammoniated silver are used, the section placed with continuous agitation into the first bath for 30 sec and the second bath for 60 sec. The sections are then transferred directly into a reducing bath (formalin 10%, 2ml; alcohol 96%, 5 ml; citric acid 1%, 1.5 ml and distilled water, 4.5 ml) for 2 min and from there to 5% Na₂S₂O₃ for 1 min, rinsed in 3 changes of distilled water, dehydrated and mounted.     

Differentiated Staining of Osmium Tetroxide-Fixed, Vestopal-w-Embedded Tissue in thin Sections

Tissues were fixed at 20° C for 1 hr in 1% OsO₄, buffered at pH 7.4 with veronal-acetate (Palade's fixative), soaked 5 min in the same buffer without OsO₄, then dehydrated in buffer-acetone mixtures of 30, 50, 75 and 90% acetone content, and finally in anhydrous acetone. Infiltration was accomplished through Vestopal-W-acetone mixtures of 1:3, 1:1, 3:1 to undiluted Vestopal. After polymerisation at 60° C for 24 hr, 1-2 μ sections were cut, dried on slides without adhesive, and stained by any of the following methods. (1) Mayer's acid hemalum: Flood the slides with the staining solution and allow to stand at 20°C for 2-3 hr while the water of the solution evaporates; wash in distilled water, 2 min; differentiate in 1% HCl; rinse 1-2 sec in 10% NH,OH. (2) Iron-trioxyhematein (of Hansen): Apply the staining solution as in method 1; wash 3-5 min in 5% acetic acid; restain for 1-12 hr by flooding with a mixture consisting of staining solution, 2 parts, and 1 part of a 1:1 mixture of 2% acetic acid and 2% H₂SO₄ (observe under microscope for staining intensity); wash 2 min in distilled water and 1 hr in tap water. (3) Iron-hematoxylin (Heidenhain): Mordant 6 hr in 2.5% iron-alum solution; wash 1 min in distilled water; stain in 1% or 0.5% ripened hematoxylin for 3-12 br; differentiate 8 min in 2.5%, and 15 min in 1% iron-alum solution; wash 1 hr in tap water. (4) Aceto-carmine (Schneider): Stain 12-24 hr; wash 0.5-1.0 min in distilled water. (5) Picrofuchsin: Stain 24-48 hr in 1% acid fuchsin dissolved in saturated aqueous picric acid; differentiate for only 1-2 sec in 96% ethanol. (6) Modified Giemsa: Mix 640 ml of a solution of 9.08 gm KH₂PO₄ in 1000 ml of distilled water and 360 ml of a solution of 11.88 gm Na₂HPO₄-2H₂O in 1000 ml of distilled water. Soak sections in this buffer, 12 hr. Dissolve 1.0 gm of azur I in 125 ml of boiling distilled water; add 0.5 gm of methylene blue; filter and add hot distilled water until a volume of 250 ml is reached (solution “AM”). Dissolve 1.5 gm of eosin, yellowish, in 250 ml of hot distilled water; filter (solution “E”). Mix 1.5 ml of “AM” in 100 ml of buffer with 3 ml of “E” in 100 ml of buffer. Stain 12-24 hr. Differentiate 3 sec in 25 ml methyl benzoate in 75 ml dioxane; 3 sec in 35 ml methyl benzoate in 65 ml acetone; 3 sec in 30 ml acetone in 70 ml methyl benzoate; and 3 sec in 5 ml acetone in 95 ml methyl benzoate. Dehydrated sections may be covered in a neutral synthetic resin (Caedax was used).     

Impregnation of Oligodendroglia in Nervous Tissue Kept in Formalin for Many Years

A method for impregnating oligodendroglia in nervous tissue (monkey) fixed and preserved in formalin for many years is described. This tissue is reconditioned by placing 12 to 30μ frozen sections of it in concentrated ammonia (sp. gr. 0.90) and by washing them slowly for 24 hours with a 1 mm. stream of water. The fluid is then poured off the sections; the jar is refilled with concentrated ammonia; and washing is repeated for another 24 hours. The sections are then plunged into concentrated ammonia for 7 minutes.

After treatment in ammonia, the sections are incubated for one hour at 38^oC. in Globus' 5% hydrobromic acid solution. They are washed again, in distilled water, and then impregnated in a “medium” strength ammoniacal silver carbonate solution (5 ml. of 10% AgNO₃ added to 15 ml. of 5% Na₂CO₃. The precipitate is dissolved in concentrated ammonia and diluted to SO ml. with distilled water). Impregnation is followed by reduction in 1% formalin without agitation; fixation in 5% Na₂S₂O₃; dehydration, and mounting in clarite.

Typical oligodendroglia (Fig. 1) were made visible by use of the method outlined in this paper.     

Histochemical Demonstration of Pyrophosphatase

Fresh tissue slices were fixed in 5% formalin containing 0.9% NaCl for 10-20 min and frozen sections therefrom floated for 3 hr at 37°C on an incubating mixture made as follows. Sodium pyrophosphate (Na₄P₂O₇-12H₂O), 1.088 gm was dissolved in 20-30 ml of distilled water and to this was added ferric chloride (FeCl₃-6H₂O), 0.61 gm dissolved in 10-15 ml of water. The precipitate was just dissolved by cautiously adding 5-10% aqueous Na₂CO₃ solution and the pH adjusted to 7.2 with 1N HCl. The volume was made up to 100 ml and 0.9 gm of NaCl added. Before use, 1 ml of 10% Mg(NO₃) was added. After incubation, sections were washed 10-15 min in 0.9% NaCl, then mounted on glass slides and air-dried. When dry, the slides were immersed in 0.9% NaCl containing 0.2-0.5% ammonium sulfide for 2-3 min, then dehydrated rapidly through graded alcohols, cleared, and covered in balsam. Sites of pyrophosphatase activity stained in various shades of green. Acid pyrophosphatase also was histochemically demonstrated by the same principle, excepting that the substrate solution was adjusted to pH 3.7-4.0 with acetate buffer. The pattern of distribution of pyrophosphatase and glycerophosphatase was almost identical.     

A Controllable Silver Stain for Nerve Fibers and Nerve Endings

A paraffin section method is described with a yellow-brown-black color range comparable to that of Ranson's pyridine silver block stain. After impregnation with activated protargol and reduction with a fine grain photographic developer, silver nitrate impregnation and reduction are repeated as often as necessary. The procedure is as follows:

Place hydrated sections of tissue fixed in chloral hydrate (25 g. in 100 ml. of 50% alcohol) in 1% aqueous protargol (Winthrop Chemical Co.) containing 5-6 g. metallic copper for 12-24 hours. After rinsing in 2 changes of distilled water, reduce 5 to 10 minutes in: Elon (Eastman Kodak Co.) 0.2 g., Na₂SO₃, dessicated, 10 g., hydroquinone 0.5 g., sodium borate powder 0.1 g., distilled water 100 ml. Wash thoroly in 4 or 5 changes of distilled water and place in 1% aqueous AgNO₃ for 10-20 minutes at 28°-50° C. Rinse in 2 or 3 changes of distilled water and reduce in the elon-hydroquinone solution. After thoroly washing in 4 or 5 changes of distilled water, examine under microscope.

If too pale, treat again in silver nitrate for 10-20 minutes, rinse, reduce 5-10 minutes and wash thoroly until nerve fibers show distinct microscopic differentiation, then dehydrate, clear and mount.     

1. Effects of Embedding 2. Experiments with Modifications

Paraffin embedding was found to be satisfactory for brain stained by a modification of the Golgi dichromate-silver method. Nitrocellulose embedding caused fading in a few specimens. Several modifications in which the tissue was impregnated with silver nitrate before treating it with potassium dichromate were investigated. The following one is recommended. Fix pieces of brain 5-6 mm. thick for 2 days in: silver nitrate;0.5%, 90 ml.; formalin, comml. unneutralized (37-40% gas), 10 ml.; pyridine, pure, 0.05-0.1 ml. Mix in the order given and test for pH with brom cresol purple. A pH of 5.5-6.0 is about optimum and the amount of pyridine added can be varied to adjust it. A slight turbidity of the fixing fluid may be disregarded, but precipitation indicates too much alkalinity. Rinse the tissues with distilled water and place them in a mixture of potassium dichromate, 2.5%, 100 ml. and osmic acid, 1%, 1 ml., for 3-5 days. Wash in water, dehydrate with alcohol and embed in soft paraffin for thick sectioning. Greater intensity of staining (but with an increase in precipitate) can be secured by rinsing the blocks after the dichromate treatment and resilvering in a 0.5% solution of silver nitrate for a day or two, then washing, dehydrating and embedding. This modification of the Golgi method was worked out on brain of adult rat, guinea pig, cat and monkey. Results with fetal material were not good. All solutions used were aqueous, and staining was done at room temperature.     

A Tetrachrome Stain for Fresh, Mineralized Bone Sections, Useful in the Diagnosis of Bone Diseases

Fresh, unprocessed bone is ground to sections 75-100 μ thick, stained in an aqueous solution composed of fast green FCF, 0.1 gm; orange G, 2.0 gm; distilled water, 100.0 ml; and adjusted to pH 6.65, then in a mixture of 1 part alcoholic solution of 0.25% celestine blue B and 9 parts of alcoholic solution of 0.1% basic fuchsin. Surface stain is removed by grinding sections to 50 μ and washing them in 1% invert soap (Zephiran) to remove adherent debris. (Commercial detergents and alkaline soaps may interfere with chromophore groups of the dyes.) Wash in tap water; rinse in distilled water and differentiate in 1% acetic alcohol. Dehydrate in ascending alcohols, clear in xylene and mount permanently in a neutral, synthetic resin. Active osteoid seams stain dark to light green; resting osteoid seams, red to bright orange red; transitional osteoid seams, geenish-yellow, orange red to red; older, partly mineralized matrix, orange; new, partly mineralized matrix, red; osteocyte nuclei, red; osteoblasts and osteoclasts, greenish-blue to dark purple nuclei and green or light green cytoplasm. Hyper-trophic and differentiating cartilage cells are stained light pink and dark red respectively. The staining reactions are consistent; the solutions are stable.     

1. Effects of Embedding 2. Experiments with Modifications

Paraffin embedding was found to be satisfactory for brain stained by a modification of the Golgi dichromate-silver method. Nitrocellulose embedding caused fading in a few specimens. Several modifications in which the tissue was impregnated with silver nitrate before treating it with potassium dichromate were investigated. The following one is recommended. Fix pieces of brain 5-6 mm. thick for 2 days in: silver nitrate;0.5%, 90 ml.; formalin, comml. unneutralized (37-40% gas), 10 ml.; pyridine, pure, 0.05-0.1 ml. Mix in the order given and test for pH with brom cresol purple. A pH of 5.5-6.0 is about optimum and the amount of pyridine added can be varied to adjust it. A slight turbidity of the fixing fluid may be disregarded, but precipitation indicates too much alkalinity. Rinse the tissues with distilled water and place them in a mixture of potassium dichromate, 2.5%, 100 ml. and osmic acid, 1%, 1 ml., for 3-5 days. Wash in water, dehydrate with alcohol and embed in soft paraffin for thick sectioning. Greater intensity of staining (but with an increase in precipitate) can be secured by rinsing the blocks after the dichromate treatment and resilvering in a 0.5% solution of silver nitrate for a day or two, then washing, dehydrating and embedding. This modification of the Golgi method was worked out on brain of adult rat, guinea pig, cat and monkey. Results with fetal material were not good. All solutions used were aqueous, and staining was done at room temperature.     

An improved silver stain for developing nervous tissue.

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.1% formol as reducing agent, 2.5% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

An Improved Silver Stain for Developing Nervous Tissue

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.l% formol as developed agent, 25% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

Silver Impregnation of Nerve Fibers in Teeth After Decalcification With Ethylenediaminetetraacetic ACID

The difficulties in impregnating bony tissues, which occur after decalcification with acids or electrolysis are avoided by decalcification with ethylenediaminetetraacetic acid at pH 8.2-8.5. The decalcification of adult human teeth which have been cut to a thickness of 2-5 mm takes 1-2 mo. If frozen sections of the decalcified teeth are impregnated 24 hr in 20% AgNo₃, rinsed through 6 changes of 20% neutralized (CaCO₃) formalin, blotted thoroughly with a cloth and placed in an ammoniated silver solution for 15-20 min, reliable impregnation of nerve fibers is obtained. The stock ammoniated silver solution is prepared by adding concentrated NH₄OH to 10-20 ml of 20% AgNO₃ until the precipitate formed by it is dissolved and then adding a few drops of the silver solution until the first permanent opalescence of the mixture is obtained. From this 2 ml are diluted directly before use with 6 ml of distilled water and 4 drops of concentrated NH₄OH added. The diluted stock solution should be used for few (5-10) sections only. The rest of the technic is done in the routine manner.     

Alcoholic Bouin fixation of insect nervous systems for bodian silver staining. III. A shortened, single impregnation method

Satisfactory Bodian silver staining of paraffin wax sections of both locust (Schistocerca gregaria) and cockroach (Periplaneta americana) central nerve tissue can be obtained with only one impregnation, instead of the usual two, by the following modified procedure. Freshly dissected ganglia are fixed in an improved synthetic alcoholic Bouin (40% formaldehyde 0-15: ethanol 25: acetic acid 5: picric acid 0.5: either ethyl acetate 5 and diethoxymethane 15, or ethyl acetate 25: distilled water to 100). Formaldehyde content governs intensity of glial staining (little or none without formaldehyde) and the mixture with more ethyl acetate substituted for diethoxymethane gives more intense staining overall. Sections are impregnated once only, overnight, in 2% Protargol solution brought to about pH 8.4 with ammonium hydroxide and containing 1.3 g of copper per 65 ml. Depending on fixative composition, species, section thickness and contrast desired between nerve fibers and background, the subsequent distilled water rinse is shortened or omitted and sections are developed in 1% hydroquinone with sodium sulfite content reduced (to 2.5-4% Na2SO3.7H2O) for thinner (10 micrometer) sections but normal (10%) for thicker (20 micrometer) ones. Sections are finally washed, gold intensified, treated with sodium thiosulfate and dehydrated, cleared and mounted as usual. Results are slightly lighter than with normal double impregnation but entirely suitable for studies of neuroanatomy.     

A Copper Sulfate-Silver Nitrate Method for Nerve Fibers of Planarians

For staining in toto, planarians are fixed in a mixture of 10 ml of commercial formalin, 45 ml of 95% ethanol and 2 ml of glacial acetic acid. After treatment with 70% ethanol 3-10 days, they are washed in distilled water and immersed in 10% CuSO₄. 5H₂O for 3 hr at 50° C, transferred without washing to 1% AgNO₃ for 1.0-1.5 hr at 50° C; and then developed in: 10 ml of 1% pyrogallol, 100 ml of 56% ethanol and 1 ml of 0.2% nitric acid. Gold toning, 5% Na₂S₂O₃ and dehydration follow as usual. For staining sections, material is fixed in the same fixative, embedded in paraffin and sectioned at 10 μ. After bringing sections to water, they are immersed in 20% CuSO₄. 5H₂O for 48 hr at 37° C; then rinsed briefly in distilled water and placed in 7% AgNO₃ for 24 hr at 37° C. They are washed briefly in distilled water and reduced in: hydroquincne, 1 gm; Na₂SO₃, 5 gm and distilled water 100 ml. Gold toning, followed by 5% Na₂S₂O₃ and dehydration completes the process. Any counterstaining may follow.     

The Use of Protein Silver for Staining Protozoa

When commercially prepared silver products suitable for staining protozoa by the Bodian silver technic apparently became unavailable, a substitute for Protargol was prepared as follows: 0.9 g. of gelatin is dissolved by heat in 100 ml. of distilled water; to this 0.1 g. of silver nitrate is added at 60°C; this solution is poured into Columbia staining dishes (10 ml.) in which one or two drops of M/10 sodium hydroxide have been added. Copper is not used in the impregnating bath. Smears fixed in Hollande's or Schaudinn's fixatives are bleached and impregnated for 36 hours or more at 35°C. Impregnated smears are reduced with a mixture of hydroquinone and sodium sulfite, and toned with gold chloride as recommended by Kirby (1945).     

Staining Neuroglia with Silver Diammino-Hydroxide after Sensitizing with Sodium Sulfite and Embedding in Paraffin

Pieces of brain are fixed in formalin ammonium bromide for about 4 days at room temperature, and given the following treatment: After washing, dehydrating and clearing, embed in paraffin, section and mount. Deparaffinize sections and pass through graded alcohols to water. Sensitize in 5% sodium sulfite for 2 hours, wash in distilled water and impregnate with silver diamminohydroxide solution 2-5 minutes at room temperature. Reduce in 2% formalin, wash in distilled water and tone in gold chloride. Fix in 5% hypo, counterstain with 1% picric acid, dehydrate and cover in balsam. Equally good results are obtained by impregnating with Hortega's strong silver carbonate. The microglia, oligodendroglia, fibrous and protoplasmic astrocytes in the cat, rabbit, newborn and adult human are successfully stained by this method.