Triple Silver Impregnation for Selective Staining of Avian Nerves

Frozen sections of avian tissue fixed 7 days or longer in 10% formalin or formol-saline are cut at 20-50 μ, left in distilled water for 2 hr, and placed in 0.002% aqueous AgNO₃ for 3-4 days. Subsequent procedure is essentially that of Weddell and Glees. Sections are placed in 20% AgNO₃ for 30 min, then carried through 3 baths of 3% formalin in less than 10 min. Immediately thereafter they are washed 1-2 sec in a 0.1% solution of NH₄OH (cone) and placed in the ammoniacal silver solution (made with 20% AgNO₃) until the nerves become distinct, as seen under a microscope; usually, in about 15 min. After washing briefly, the sections are fixed in 5% Na₂S₂O₃ for 3-10 min, dehydrated, cleared, and mounted in the usual way.     

Staining Formalin-Fixed Nerve Tissue with Mercuric Nitrate

Experiments were made to test the impregnating effect of Hg(NO₃)₂ on nervous tissue that had been fixed and chromated with solutions of known pH. Brains of cats, kittens, rats and mice were fixed by the pulsating-perfusion method of Haushalter and Bertram (1955), after first washing out the blood with saline-acacia solution, at pH 7.0, then followed by a 10% formol-saline-acacia fixative of the same pH. The removed brains were sliced to 3 mm thickness and further fixed 1-2 days in 10% formalin whose pH was also adjusted to 7.0. Chromation with acidified ZnCrO₄ at pH 3.1 for 1 day followed by impregnation for 2 days in a saturated solution of Hg(NO₃)₂ at pH 5.5-6.0 effected the staining. Dehydration, paraffin embedding and sectioning completed the process. Some moderately successful stains were made with mercuric salts with no chromation, but it was found that fixation at pH 7.0-7.2 followed by chromation at pH 3.1, and later by impregnation in Hg(NO₃)₂ at pH 5.8-6.0 was optimum for best staining of nerve cells for their processes. The advantages of the technique are: (1) selective staining of nerve cells, especially the axonic details; and (2) a relatively short time needed for its completion.     

A Chlorate-Formaldehyde Modification of the Golgi Method

Pieces of fresh nervous tissue 3-5 mm thick are put into a mixture of: 6% K₂Cr₂O₇, 40 ml; 5% KClO₃, 20 ml; 20% chloral hydrate, 30 ml; and concentrated formalin (38% HCHO), 10 ml; allowed to fix 3 days, with a daily change of fluid; transferred to 3% K₂Cr₂O₇ for 3 days, with twice daily changes; then to 1% AgNO₃ for 3 days at 20-25° C. Frozen sections are cut, dehydrated, cleared and mounted in Permount with a cover glass. The method gives good results for microglia and oligodendroglia in addition to the usual staining of nerve cells and their processes.     

Ammoniated Silver Carbonate for the Demonstration of Lysosomes

Hortega's ammoniated silver carbonate method was used to demonstrate lysosomes in the central nervous system and kidney of adult rats. Formol-CaCl₂, (10%:1%) fixed, frozen sections were impregnated for 10 min in Hortega's solution: 30 ml of 10% AgNO₂ and 90 ml of 5% Na₂CO₃, with concentrated NH₄OH added until the precipitate dissolved, then distilled water to make 400 ml. This procedure revealed silver-positive cytoplasmic structures whose form, shape and distribution were similar to that seen by staining adjacent sections for acid phosphatase. A short fixation of 18-24 hr appears to be essential. A useful, nonenzymatic method for the demonstration of lysosomes is thereby available.     

A Silver Impregnation Method for Nervous Tissue Suitable for Routine use with Mounted Sections

A simple, reliable silver impregnation method for nervous tissue is described for tissues fixed in various fixatives including formalin, Bouin, and Sum. Sections are impregnated in a solution containing 1 g Protargol, 2 ml of a 1% Cu(NO₃)₂ solution, 2 ml of a 1% AgNO₃ solution, and 2-4 drops 30% H₂O₂ in 100 ml distilled water. Sections are impregnated 4-5 days at 37 C and thereafter reduced in a hydroquinone-formalin solution. This is followed by gold toning and subsequent reduction, dehydration and mounting. This method has been found to be very reliable and selective.     

The Application of Del Rio Hortega'S Silver Method to Epon-Embedded Tissue

OsO-fixed, Epon- or Araldite-embedded tissue can be stained by the silver amminocarbonate method of del Rio Hortega. Sections, 0.5-1.5 μ thick, are floated on the silver solution and impregnated for 0.5-2 hr at 60 C. Pyridine, 1-2 drops/10 ml of staining solution, facilitates the staining. Reduction in 1% formalin and fixation by Na₂S₂O₃ are optional. Staining may be influenced by the buffering vehicle in the fixative and appears to be dependent on sites of osmium binding. Cytoplasmic processes and basement membranes are well stained, as are intracytoplasmic organelles. Nuclear and nucleolar staining is variable.     

Marchi's Staining Method: Studies of Some of the Underlying Mechanisms Involved

Spinal cord of cat and rabbit was stained, after experimental lesions, by variations of Marchi's method. The following conclusions were drawn:

1. The presence of an oxidizing agent (K₂Cr₂O₇, NaIO₃, or KCIO₃) in the osmic acid solution is of primary importance and a preliminary oxidation in Mueller's fluid is unnecessary or even detrimental.

2. Acetic acid added to Marchi's fluid, accentuates the action of the oxidizing agent in restraining the staining of normal myelin.

3. Too high concentration of oxidizing agent or of acid may inhibit staining of degenerating myelin.

4. Marchi's and Busch's methods have been modified as follows: Fix one day in 10% formalin and transfer without washing to the staining mixture, either A or B. Staining mixture A: Marchi's fluid plus 1 to 3% glacial acetic acid. B: An aqueous solution containing KCIO₃ 0.25%, osmic acid 0.33%, and acetic acid 1%. Stain about one week. These methods worked on spinal cord and medulla, but cannot be recommended for brain.

5. The detrimental effects of long post mortem autolysis or of prolonged fixation in formalin may be counteracted to some degree by increasing the concentration of the acid in Marchi's fluid up to 5% or of the KCIO₃ up to 0.4% in the modified Busch's fluid.     

Correlation of CrystaL Growth with the Staining of Axons by the Golgi Procedure

The surface of the specimens subjected to a modified Golgi technique (formalin fixed material; specimens in the following solution for 8-10 days at 27 C: 3% K₂Cr₂O₇, 100 ml, with the addition of 2.5-10 ml of 10% formalin and 6-25 gm of sucrose; then in 0.75% AgNO₃ for at least 2 days at 27 C) is sometimes covered with a fur of filamentous crystals and sometimes with a powdery precipitate of laminar crystals. In a series of experiments in which about 500 blocks of tissue were treated with variations of the staining procedure, good axonal stain was positively correlated with the appearance of filamentous crystals. These filaments have a thickness of 1-4 μ and grow at a rate of 160-330 μ/hr, reaching a length of 2-7 mm.     

Metachromatic Reaction of Pancreatic B Cells to Toluidine Blue; Influence of pH on Staining

The granules of islet B cells show an intense β metachromasia when paraffin sections of pancreas fixed in Bouin's fluid or formalin are dipped for 1 min in a 0.1% aqueous solution of toluidine blue O₂ buffered to pH 6.0 with acetate or phosphate. This reaction provides a quick method for surveying the condition of B cells in experimental work. A weak staining is observable at pH 4.5 and becomes distinct at pH 5.5-6.0. Oxidation of sections (0.25% KMnO₄ in 0.5% H₂SO₄, for 1 min, recommended) prior to staining intensifies the metachomatic reaction conspicuously. The metachromatic substance could not be demonstrated after fixation in either ethanol or acetone. It corresponds to the aldehyde fuchsin-positive and pseudoisocyanin-metachromatic substance in its occurrence and distribution in the B cells, as shown by different physiological states of various animals, including fasted and glucose-administered guinea pigs. It is thought to be topographically coincident but not necessarily identical to insulin.     

Staining of Oligodendroglia with Silver Ammino Tungstate in Unembedded and Embedded Material

Specimens of brain or spinal cord fixed in formalin, Cajal's formol-bromide, or Koenig, Groat and Windle's formalin-acacia can be used to stain oligodendrocytes in frozen, in paraffin, or in celloidin sections. The sections are soaked 3-5 min in 0.02% acetic acid, pH 3.4, then rinsed 2-3 sec in 3% H₂O₂ and transferred to a silver bath prepared as follows: Mix equal parts of 10% AgNO₃ and 10% Na₂WO₄, and dissolve the precipitate with concentrated NH₄OH; avoid an excess of ammonia. Silver at room temperature for 15-20 sec, develop in 1% formalin, dehydrate, and mount. For embedded material, prepare a mixture consisting of 1 part of 10% aqueous Aerosol MA and 4 parts of 10% Aerosol OT in 95% alcohol. Add 5 drops of this mixture to each 50 ml of dilute acetic acid and 3% H₂O₂; 5 drops to each 20 ml of the silver bath.     

A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

Extensive experimentation with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections IS to 25 μ, frozen sections 25 to 40 μ. Place sections for 24 hours in 50% alcohol to which 1% by volume of NH₄OH has been added. Transfer the sections directly into a 1% aqueous solution of protargol, containing 0.2 to 0.3 g. of electrolytic copper foil which has been coated with a 0.5% solution of celloidin, and allow to stand for 6 to 8 hours at 37° C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24 to 48 hours at 37° C. directly into a pyridinated solution of alcoholic protargol (1.0% aqueous solution protargol, 50 ml.; 95% alcohol, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated copper. Rinse briefly in 50% alcohol and reduce 10 min. in an alkaline hydroquinone reducer (H₃BO₃, 1.4 g.; Na₂SO₃, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroly in water and tone for 10 min. in 0.2% aqueous gold chloride, acidified with acetic acid. Wash in distilled water and reduce for 1 to 3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3 to 5 min. with 5% aqueous Na₂S₂O₃+5H₂O. Wash in water and stain overnight in Einarson's gallocyanin. Wash thoroly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock solution, 20 ml.; distilled water, 30 ml.) of the following stock staining solution: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid, 8 ml.) and stain for 1 hour. Differentiate with 70% and 95% alcohol; pass the sections thru butyl alcohol and cedar oil; mount.     

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.     

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.     

Selective Staining of Neurosecretory Material in Semithin Epoxy Sections by Gomori's Aldehyde Fuchsin

The epoxy resin was removed from semithin (1 μm) sections by immersing them for 30 sec in sodium methoxide (Mayor et al., J. Biophys. Biochem. Cytol., 9: 909-10, 1961) and then processed as follows: (1) left for 1-3 hr at 60 C in a mixture of formalin, 25 ml; glacial acetic acid, 5 ml; CrO₃, 3 gm; and distilled water, 75 ml: (2) oxidized 10 min in a 1:1:6 v/v mixture of 2.5% KMnO₄, 5% H₂SO₄ and distilled water: (3) bleached in 1% oxalic acid, and (4) stained for 15 min in aldehyde fuchsin, 0.125% in 70% alcohol, or in a 1% aqueous solution of toluidine blue. The neurosecretory material is selectively stained.     

Acceleration of Mallory's Phosphotungstic Acid-Hematoxylin Staining for Skeletal Muscle Fixed in Formalin

The staining time for mammalian skeletal muscle fixed in neutral phosphate-buffered formalin was shortened from 12-24 hr to 10-30 min. The permanganate-oxalate sequence was omitted although oxidation by periodic acid or with iodine was found to be necessary. The material was embedded in paraffin and cut 6 μ or less. Deparaffinized sections were treated with 1% alcoholic iodine for 10 rain followed by 5% Na₂S₂O₃ for 2 min and placed in an oven at 60 C for 10-30 min to stain in a preheated mixture of 50 ml of ripened Mallory's phosphotungstic acid-hematoxylin and 1 ml of 2% phosphomolybdic acid. Experiments with fixation showed that the staining procedure followed Zenker's fluid successfully but not Bouin's fluid. Oxidation by KMnO₄ was effective only after Zenker fixation; oxidation by CrO₃ was unsuccessful.     

Staining Tissue of the Central Nervous System with Luxol Fast Blue and Neutral Red

The tissue is fixed in 10% neutral saline formalin for 1 day to 3 wk depending on the size of the block, dehydrated and embedded in paraffin. The sections are stained at 57° C for 2 hr, then at 22° C for 30 min, in a 0.0125% solution of Luxol fast blue in 95% alcohol acidified by 0.1% acetic acid. They are differentiated in a solution consisting of: Li₂CO₃, 5.0 gm; LiOH-H₂O, 0.01 gm; and distilled water, 1 liter at 0-1° C, followed by 70% alcohol, and then treated with 0.2% NaHSO₃. They are soaked 1 min in an acetic acid-sodium acetate buffer 0.1 N, pH 5.6, then stained with 0.03% buffered aqueous neutral red. Sections are washed in distilled water, 1 sec, then treated with the following solution: CuSO₄·5H₂O, 0.5 gm; CrK(SO₄)₂·12H₂O, 0.5 gm; 10% acetic acid, 3 ml; and distilled water, 250 ml. Dehydration, clearing and covering complete the process. Myelin sheaths are stained bright blue; meninges and the adventitia of blood vessels are blue; red blood cells are green. Nissl material is stained brilliant red; axon hillocks, axis cylinders, ependyma, nuclei and some cytoplasm of neuroglia, media and endothelium of blood vessels are pink.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

A HYDROXYL-DEPENDENT SILVER METHOD FOR NERVE AXOPLASM

Axoplasm is selectively impregnated by the following steps: (1) fixation in 10% formalin or in 10% formalin with added sucrose, 15%, and concentrated NH₄OH, 1%, for 1-7 days; (2) frozen sections; (3) extraction of the sections in 95% ethyl alcohol, absolute alcohol, xylene, and 95% ethyl alcohol and absolute alcohol, 1 hr each; (4) distilled water, 3 changes of 10 min each; (5) 20% AgNO₃ (aq.) at 25°C, 30 min; (6) distilled water, 3 changes of 1-2 sec each; (7) 6.9% K₂CO₃, 1 hr; (8) water, 3 changes of about 1 min each; (9) 0.2%AuCl₃, 2 min; (10) distilled water; (11) 5% Na₂S₂O₃, 2 min; (12) washing, clearing and mounting. This procedure is proposed as a simplified stain for axoplasm, with other tissue components remaining unstained. The few reagents necessary suit this method for histochemical investigation of the mechanism of silver staining.     

Effect of Fixatives on Silver Impregnation of Plant Cells

The kind of fixative and duration of fixation modify the affinity of plant cell structures, as shown by a 10-15 hr impregnation at 70 C in 2% aqueous AgNO₂, and a 1-2 hr reduction at room temperature by a 1:1 mixture of 10% formalin and 1% hydroquinone. Cytoplasmic staining was enhanced by fixing in salts of heavy metals, in buffered 6.5% glutaraldehyde, and in 0.5% picric acid. Nuclear staining was prominent after mixtures of glutaraldehyde and hydroquinone, after formalin and pyrogallol, and after acetone, propylene glycol or ether. Nucleolar staining was favored by fixing in 10% formalin, in 5% formalin containing 0.5% hydroquinone, in 50% ethanol containing 0.5% pyrogallol, or in ethylene glycol. Chromosome staining was favored by fixation in 50% acetic or propionic acid, in 2% trichloroacetic acid, and in methanol or ethanol. The best morphological preservations were seen after 50% acetic acid, 6.5% glutaraldehyde, or the 5% formalin-0.5% hydroquinone mixture.     

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.