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).     

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.     

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.     

Luxol Fast Blue Arn: A New Solvent Azo Dye with Improved Staining Qualities for Myelin and Phospholipids

Luxol fast blue ARN (Du Pont, C.I. solvent blue 37) is a diarylguanidine salt of a sulfonated azo dye. This dye was compared with other Luxol blue and Luxol black dyes. Luxol fast blue ARN has improved staining qualities for phospholipids and myelin, and can advantageously be substituted for Luxol fast blue MBS (MBSN). Appropriate staining times for a 0.1% dye solution in 95% ethanol (containing 0.02% acetic add) at 35°-40° C range from 2-3 hr. After staining, the sections should be rinsed in 95% ethanol, rinsed in distilled water, and differentiated for 2 sec in 0.005% Li₂CO₃, rinsed in 70% ethanol, washed in water, and counterstained as required. Phospholipids and myelin selectively stain deep blue. A fixative containing CaCl₂, 1%; cetyltrimethylammonium bromide, 0.5%; and formaldehyde, 10%, in water gave excellent results with brain. However, 10% formalin can be used. The staining of the phospholipids is probably due to the formation of dye-phospholipid complexes.     

A Phloxine-Azure-Hematoxylin Sequence for Differential Staining of Cells in Pancreatic Islets

Sections of 6 μ from tissues fixed in Susa or in Bouin's fluid (without acetic acid) and embedded in paraffin were attached to slides with Mayer's albumen, dried at 37 C for 12 hr, deparaffinized and hydrated. The sections fixed in Susa were transferred to a I₂-K₁ solution (1:2:300 ml of water); rinsed in water, decolorized in 5% Na₂S₂O₃; washed in running water, and rinsed in distilled water. Those fixed in Bouin's were transferred to 80% alcohol until decolorized, then rinsed in distilled water. All sections were stained in 1% aqueous phloxine, 10 min; rinsed in distilled water and transferred to 3% aqueous phosphotungstic acid, 1 min; rinsed in distilled water; stained 0.5 min in 0.05 azure II (Merck), washed in water; and finally, nuclear staining in Weigert's hematoxylin for 1 min was followed by a rinse in distilled water, rapid dehydration through alcohols, clearing in xylene and covering in balsam or a synthetic resin. In the completed stain, islet cells appear as follows: A cells, purple; B cells, weakly violet-blue; D cells, light blue with evident granules; exocrine cells, grayish blue with red granules.     

Counterstaining Golgi-Cox Impregnations with Luxol Fast Blue as a Myelin Stain

Immerse pieces of brain tissue 4 wk in solutions A and B, mixed just before use: A. K₂Cr₂O₇, 1 gm; HgCl₂, 1 gm; boiling distilled water, 85 ml. Boil A for 15 min, cool to 2 C and add: B. K₂CrO₄, 0.8 gm; Na₂WO₄, 0.5 gm; distilled water, 20 ml. Rinse in water and immerse 24 hr in LiOH, 0.5 gm; KNO₃, 15 gm; distilled water, 100 ml. Wash 24 hr in several changes of 0.2% acetic acid and then for 2 hr in tap water. Dehydrate and embed in celloidin. Process a 60 μ section through 70 and 95% ethanol, a 3:1 mixture of absolute ethanol and chloroform, and toluene. Immerse it for 5 min in a solution containing methyl benzoate, 25 ml; benzyl alcohol, 100 ml; chloroform, 75 ml. Orient the section on a chemically clean slide and let air-dry 5-10 min. Process through toluene, 3:1 ethanol-chloroform and 95% ethanol. Place the section for 5-60 min at 60 C in a solution made up of: Luxol fast blue G (Matheson, Coleman and Bell), 1 gm; 95% ethanol, 1000 ml; 10% acetic acid, 5 ml. Hydrate to water and immerse in 0.05% Li₂CO₃ for 3-4 min. Differentiate in 70% ethanol and place in water. Immerse for 5-15 min in a mixture of two solutions: A. cresylechtviolet (Otto C. Watzka, Montreal), 2 gm; 1 M acetic acid, 185 ml; B. 1 M sodium acetate, 15 ml; distilled water, 400 ml; absolute ethanol, 200 ml. Dehydrate to 3:1 ethanol-chloroform. Clear in toluene and apply a coverslip. The technique produces fast Golgi-Cox impregnated neurons against a background of counterstained myelinated fibers. Patterns of the myelinated fibers can be used to localize impregnated neurons.     

Bromphenol Blue as A Stain for Muscle Striations

Sections from formalin-fixed muscle are stained 4-24 hr with a 0.05% solution of bromphenol blue in 2% acetic acid, rinsed with water and placed in 0.5% acetic acid for 5-10 min. They are then treated 30 sec with tap water substitute (KHCO₃, 0.2 gm; MgSO₄, 2 gm; distilled water, 100 ml), rinsed, dehydrated in alcohol, cleared in xylene and covered in a polystyrene mountant Striatums of both cardiac and skeletal muscle fibers are fully resolved under oil immersion, against the blue background of the other parts of the fibers.     

Staining Residual Lipids in Ultrathin Sections of Tissues Embedded in Polyester Resin

Rat suprarenal glands fixed in Palade's 1% OsO₄, buffered at pH 7.7 with veronal-acetate, to which 0.1% MgCl₂ was added, were embedded in Vestopal-W and sectioned at 0.2-1 µ. The sections were attached to slides by floating on water, without adhesive, and drying at 60-80° C, placed in acetone for 1 min and then treated with the following staining procedure: Place the preparation in a filtered solution of oil red O, 1 gm; 70% alcohol, 50 ml; and acetone, C.P., 50 ml; for 0.5-1 hr. Rinse in absolute ethyl alcohol; drain; counterstain with 0.5% aqueous thionin for 5 min; rinse in distilled water; drain; stain in 0.2% azure B in phosphate buffer at pH 9, for 5 min. Dry and apply a drop of immersion oil directly on the section. The preparations are temporary. Ciaccio-positive lipids, rendered insoluble by OsO, fixation, stained red to ochre.     

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.     

A Rhodocyan Technic for Staining the Anterior Pituitary

A reproducible, one-step, differential staining technic which uses routine formalin-fixed tissue and gives brilliantly contrasting results is produced by incubating sections for 1 hr in a 60° C oven in the following dye mixture: 1% eosin B (CI#771), 8 ml; 1% anilin blue (CI#707), 2 ml; and buffer solution (0.1M citric acid, 1.1 ml; 0.2M Na₂HPO₄, 0.9 ml; distilled water, 28.0 ml) at pH 4.5. No differentiation is necessary. The method can be modified for duodenal enterochromaffin cells and alpha cells of pancreatic islets by adjusting the buffer to pH 3.6 and staining for only 3 min at 60° C.     

Staining and Mounting Helminths

The following procedure is recommended: Fix ces-todes and trematodes (while held flat between glass slides) 0.5-2.0 hr. in the following mixture: formalin, 15; acetic acid (gl.), 5; glycerol, 10; 95% ethyl alcohol, 24; distilled H₂O, 46; all proportions by volume. After freeing them from the slides, wash thoroughly in running water and stain immediately thereafter. Stock staining solution: ferric ammonium alum (violet cryst.), 2 g.; distilled H₂O (cold) 100 ml.; after solution, add 2 ml. concentrated H₂SO₄, bring to a boil; add 1 g. coelestin blue B (Nat. Aniline), boil 3-5 min.; cool and add 10 ml. absolute methyl alcohol and 10 ml. glycerol. Dilute 1 vol. with 3 vol. distilled H20 for use. Stain 5-30 min., depending on size of specimens. Wash with 2 changes 0.5 hr. each of distilled H₂O, then 50% isopropyl alcohol 12-16 hr., 50% isopropyl alcohol 2 hr., followed by graded isopropyl alcohol for dehydration. Ether: ethyl alcohol (equal parts), 1 hr., is followed by embedding in celloidin in a sheet just thick enough to cover the specimens. Trim embedded specimens and dehydrate with isopropyl alcohol, 80%, 90% and absolute. Clear in beechwood creosote. Mount in balsam with cover glasses that overlap the edges of the celloidin 1-2 mm. While drying at 37°C, refill edges of mount with fresh balsam as needed. When dry, remove excess balsam and ring the edges with ordinary gloss enamel paint.     

Staining Procedures for Ultrathin Sections of Tissues Embedded in Polyester Resin

Tissues were fixed for 30 min In cold (0-2° C) 1% OsO₄ (Palade) buffered at pH 7.7, to which 0.1% MgCl₂ was added. Dehydration was in a graded ethanol series (containing 0.5% MgCl₂) at 0-2° C, and terminated with 2 changes of absolute ethanol. Tissues were then transferred by a graded series to anhydrous acetone. Infiltration of the tissue with Vestopal-W (a polyester resin), is gradual with the aid of graded solutions of Vestopal-W in acetone. The infiltrated tissue is encapsulated and initial polymerization is done under ultraviolet light at room temperature for 8-16 hr. This is followed by final hardening at 60° C for 36-48 hr. Sections (0.2-1 μ) were cut, dried on slides, placed in acetone for 1 min and then treated by either of the following staining procedures: (1) Thionin-azure-fuchsin staining: Flood the preparation with 0.2% aqueous thionin and heat to 60-80° C for 3 min; if the preparation begins to dry, add stain. Rinse in distilled water. Flood the slide with 0.2% azure B in phosphate buffer at pH 9. Heat to 60-80° C for 3 min; do not permit the preparation to dry. Rinse in distilled water. Dip the slide in MacCallum's variant of Goodpasture's carbol-fuchsin stain for 1-2 sec. Rinse in distilled water. Check the preparation microscopically for intensity of the fuchsin stain. Repeat dips as may be needed to obtain the desired intensity. Rinse in distilled water. Dehydrate quickly in 95% and absolute alcohol; clear in 2 changes of xylene and cover in Permount or similar synthetic resin. (2) Thionin-azure counterstain for the periodic acid-Schiff reaction: Oxidize the tissue in 0.5% periodic acid for 15 min and transfer to Schiff's leucofuchsin solution for 30 min. Counterstain with 0.5% aqueous thionin for 3 min; wash in distilled water; stain in 0.2% azure B in phosphate buffer at pH 5.5; wash in distilled water; dehydrate; clear and cover as in the first method. For temporary preparations let dry after absolute alcohol and apply a drop of immersion oil directly on the section.     

Differential Staining of Gastric Glandular Cells

Fundus of stomach is fixed in 10% formalin (aqueous), Bouin's fluid or 5% trichloracetic acid (aqueous). It is embedded in paraffin, and 7μ sections are cut, mounted, deparaffinized and passed to 70% alcohol and then stained as follows: Mordant 3 min. in saturated Bismarck brown in 70% alcohol. Rinse in 70% alcohol, pass to distilled water, then overstain (2 hr.) in aniline blue, 0.5% solution in 2.5% acetic acid (aqueous). Precipitate the anilin blue with 0.5 ml. of 0.1% methyl violet solution (aqueous) dropped on die slide. Leave on 2 min. or less. Wash and differentiate in 70% alcohol. (Parietal cells dark blue). Stain 30 min. in a mixture of hematein, 0.10g.; A1C13 cryst., 0.05g.; and 70% alcohol 50 ml., prepared just before use and not filtered. Rinse in 70% alcohol and differentiate with an alcoholic extract of saffron (2 g. saffron pistils in 100 ml. 90% alcohol at 60°C. for 6 hr.) while observing the progress of differentiation microscopically. Dehydrate by dropping a 0.1 % solution of acetic acid in absolute alcohol on the section for 30 sec., followed by pure absolute alcohol, xylene, and covering in balsam.     

Azure a-Schiff, Alcian Blue, HIO4-Schiff, Naphthol Yellow S—A Sequential Staining Method for Paraffin Sections

Paraffin sections from tissue fixed 4-12 hr in 10% formalin containing 0.5% cetyl pyridinium chloride, and washed 2 hr, were stained as follows: (1) Hydrolyze in 5 N HCl at room temperature for 8.5-9 min, or use standard Feulgen hydrolysis at 60 C. (2) Stain in azure A-Schiff, 0.5% in bisulfite bleach (1 N HCl, 5; 10% Na₂S₂O₅, 5; and distilled water 90—parts by volume) for 10 min. (3) Place in bisulfite bleach 2 changes, 2 min each; wash in water, 1-2 min. (4) Stain in Alcian blue (0.1% in 0.01 2V HCl, pH 2.0) for 10 min. (5) Place in 0.01 N HCl for 2-3 min; wash in water for 1-2 min. (6) Oxidize in 0.5% HIO₄ for 5 min; wash in water, 1-2 min. (7) Stain in Schiff's leucofuchsiu, 10 min. (8) Treat with bisulfite bleach as in step 3; wash in running water, 10 min. (9) Stain in naphthol yellow S (0.01% in 1% acetic acid) for 1-2 min. (10) Place in 1% acetic acid for 2 min, dehydrate in tertiary butanol, clear and cover. Result: DNA is deep blue; acidic mucins are light blue; neutral polysaccharides, red to magenta; and proteins, yellow. Proper timing of the hydrolysis for the Feulgen reaction is the most critical step. Overhydrolysis results in green nuclei (staining by naphthol yellow S) whereas purplish nuclei are the results of insufficient hydrolysis.     

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.     

Staining Reticulin with Gold

This bromine-iodine-gold chloride-reduction sequence stains reticulin in formalin-fixed paraffin sections without risk of sections becoming detached. After hydration, sections are exposed to 0.2% bromine water containing 0.01% KBr for 1 hr, then rinsed and placed for 5 min in a solution consisting of KI, 2 gm; iodine crystals, 1 gm; and distilled water, 100 ml. After this the sections are well washed in distilled water, immersed for 5 min in 1% w/v aqueous solution of chloro-auric acid, again rinsed in distilled water, and the gold is reduced by placing in freshly made 3% H₂O₂ for 2-4 hr at 37 C, or in 2% oxalic acid for 1-3 hr at the same temperature.     

Silver Protein Staining of Nerve Fibers in Celloidin Sections

Celloidin sections from formalin-fixed brain and spinal cord of primates are stored in 70% alcohol after cutting, soaked in 2% pyridine in 50% alcohol for 6-8 hr at 37 C, and transferred to 1% concentrated NH₄OH in 50% alcohol 15-18 hr at 20-25 C. After washing and flattening, the sections are transferred to 1% silver protein solution containing 30 ml of 0.2 M H₃BO₃/100 ml. Impregnation is accomplished in 50 ml screw-top jars, 50 mm in diameter, which are filled to a depth of 35 mm, and have 1 gm of copper foil, 0.002 inch thick added. The foil is folded in loose accordion-fashion, pierced and threaded, cleaned in 5% HNO₃, rinsed in distilled water, and suspended in the solution just above the sections by fastening the thread to the jar lid. The sections are impregnated for 24 hr at 37 C, rinsed in distilled water, reduced in a solution of 5% Na₂SO₃ and 1% hydroquinone for 10 min, washed in distilled water and toned in 0.2% gold chloride for 5 min. After rinsing in distilled water, the sections are transferred to 1% oxalic acid for 45-60 sec, washed in distilled water and placed in 5% Na₂S₂O₃ for 5 min. Sections are then washed, dehydrated to 95% alcohol, cleared in terpineol, followed by 3 changes in xylene, and mounted.     

Mercuric Bromphenol Blue Staining of Precipitin Lines in Agar

Lines formed by antibody-organ antigen reactions are stained particularly well by a modification utilizing the mercuric bromphenol blue (MBB) mixture of Mazia et al. (Biol. Bull., 104: 57-67, 1953). The agar covered slides are placed overnight in 0.85% NaCI at 4 C, followed by washing for 2 hr in 0.85% NaCI at 25 C. They are then rinsed for 10 min in distilled water, and dried overnight at 37 C. The precipitin lines are fixed by immersing the slides for 25 min in 95% alcohol, followed by 5 min hydration in distilled water. They are stained for 25 min in MBB mixture (HgCI₂, 10 gm; bromphenol blue, 0.1 gm; 95% ethanol, 100 ml). Excess stain is removed by immersing in acidified alcohol (95% ethanol, 98 ml; glacial acetic acid, 2 ml). Finally, the slides are passed through alcohol and xylene, and resin-mounted under coverslips.     

Myelin Impregnation: An Improved Golgi-Cox Modification

Optic tecta of goldfish were coated with egg yolk and immersed for only one week in one of the following impregnation fluids: a) Solution A + B; A = 1 g K₂Cr₂O₇ and 1 g HgCl₂ boiled for 15 min in 85 ml distilled water and allowed to cool; B = 0.8 g K₂Cr₂O₇ and 0.5 g KWO₄ dissolved in 20 ml distilled water, b) Solution A + B two volumes diluted with boiled distilled water, c) Solution A + B four volumes diluted with boiled distilled water. Each tectum was immersed 6 hr in 100 ml distilled water containing 0.5 g LiOH and 15 g KNO₃, washed 18 hr in 500 ml 0.2% acetic acid, dehydrated with ethanol, and embedded in low viscosity nitro cellulose. Sections were cut at 100 pm with a rotary microtome after clearing with cedarwood oil. Methods b) and c) have two advantages compared with method a), the original Golgi-Cox method. First, more cells are impregnated, especially in the layers extending 200-400 μm below the surface, and dendrites as well as unmyelinated axons are well impregnated. Second, myelin sheaths are impregnated and can be recognized by their peculiar chain-like appearance. The described Golgi-Cox modification offers an appropriate method to study the morphology of superficially located nervous tissue.