A Rapid Silver-on-the-Slide Method for Nervous Tissue

A progressive silver staining method is described, which permits microscopic examination of the sections during the staining process. After formaldehyde fixation, dehydration and embedding in paraffin or celloidin, fine fibers and synaptic endings may be demonstrated. After formaldehyde fixation and mordanting in 3% K₂Cr₂O₇, myelinated fibers and mitochondria are specifically stained.

The unique feature of this method is, that the silver solution (0.5% protargol) is mixed with the reducing solution: 1.6% Rochelle salts, containing traces of Ag NO₃, MgSO₄, and K₂S (U.S.P.). The sections are placed directly into this mixture, which is then warmed to 45-55° C. Sections are removed when progressive staining is completed, washed in water, dehydrated and mounted.

In the fiber stain, nerve fibers and synaptic endings are dark brown or black, and nuclear chromatin is deep brown, against a pale yellow background. When the myelin sheath procedure is followed, the fiber bundles are deep brown, and the intensity of the staining remains the same for specific tracts, aiding in their identification.     

Glutaraldehyde-Ammoniacal Silver Carbonate-Formaldehyde Staining of Histones of Mitotic Chromosomes

Cells from monolayer culture of Chinese hamster line Don were treated by Colcemid (0.1 μg/ml) for 2 hr, trypsinized and spun; resuspended in 0.5% sodium citrate solution for 10 min, respun, and then resuspended in a small volume of the supernatant. Slide preparations were made by smearing, followed by air drying for 1 min at room temperature. They were fixed and stained by the following sequence: 2.5% glutaraldehyde in Millonig's buffer, 30 min; distilled water, 6 min, 5 changes; ammoniacal silver at 18-26 C, 10 sec; distilled water, 30 min, 5 changes; 2.5% formalin, 2 min; and distilled water, 3 changes during 15 min. Staining solution: add 225 ml of 5% Na₂CO₃ to 75 ml of 10% AgNO₃, then add concentrated NH₄OH slowly, drop by drop, until the solution is transparent. Finally add 300 ml of dstilled water. Cells treated with cold 0.25 N HCl before fixation were not stained. Sequence modifications show that chromatin does not reduce silver by itself. This method stains the sites of high histone concentrations in mitotic chromosomes of cytogenetic preparations.     

Silver staining of DNA synthesizing cells in the neural tube of chick embryos

Abstract. I report a study by light microscopy of the spinal cord of early chick embryos stained with the ammoniacal silver carbonate solution of Del Rio Hortega. Cell nuclei are stained in a selective fashion and two classes of nuclei – dark and pale – can be distinguished in the neuroepithelium. Neuronal nuclei also show a characteristic staining pattern. A radioautographic study after [³H] tyhmidine incorporation has shown that it is the dark neuroepithelial nuclei that are engaged in DNA synthesis. Dark nuclei disappear after administration of cytosine arabinoside, supporting the association between DNA synthesis and silver staining of neuroepithelial nuclei.     

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.     

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).     

A Rapid Silver Impregnation Method for Nervous Tissue: A Modified Protargol-Peroxide Technic

A rapid, reliable silver impregnation method is described for nervous tissue fixed in formol-saline, Bouin or Sum. Sections are impregnated for 10-15 minutes at room temperature or 37 C in a solution containing 0.5 g Protargol-S, 0.005-0.01 g allantoin, 1 ml of 1% Cu[NO₂]₂, 1 ml of 1% AgNO₃. and 1-2 drops of 30% H₂O₂ in 100 ml distilled water. Thereafter the dons arc reduced in a hydroquinone-formalin solution. This is followed by gold toning and subsequent reduction and mounting. Alternatively. following the first reduction, the silver image can be intensified by placing sections in a silver-allantoin bath which is followed by reduction and mounting. This method is very reliable and selective, making it suitable for general routine and research use.     

Detection of a few picograms of DNA on polyacrylamide gels by silver staining

DNA fragments separated on polyacrylamide gels are silver stained in ethanolamine solution. The staining procedure can be completed in 3 1/2 h. Illumination of the gels on a black background increases the sensitivity of detection compared with the usual transillumination. The limit level of detection is 3-5 pg per band with a cross-sectional area of 5 mm2. Five to fifty picograms of DNA may be detected quantitatively by scanning the gels. The method will detect 0.1 to 1 ng per band of low-molecular-weight RNA components.     

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.     

Adaptation of the Millon, Sudan Black B, and Periodic Acid-Schiff Technics for Block Staining of Insect Tissues

Spermatophores and reproductive systems of the beetle, Lytta nuttalli Say, fixed in Bouin's aqueous picroformol or buffered 10% neutral formol were stained in toto by the Millon, Sudan black B and periodic acid-Schiff reactions as follows. Millon: after excess fixative is removed in 70% ethanol, specimens are brought to water, stained in Millon's reagent at 60 C for 1 hr, rinsed in 2% aqueous nitric acid at 40-50 C, dehydrated rapidly, cleared, embedded and sectioned as usual. Sudan black B: specimens are taken to absolute ethanol, stained in a saturated solution of Sudan black B in absolute ethanol at room temperature for 24-48 hr, rinsed and cleared in xylene, embedded and sectioned. PAS: specimens are brought to water, oxidized in 0.5 aqueous HIO₄ at 37 C for 30 min, washed in 2 changes of water, stained in Schiif reagent at room temperature for 1 hr, rinsed in 3 changes of 0.5% aqueous potassium metabisulfite, washed in running water for 10-15 min, dehydrated, cleared, embedded and sectioned. All 3 methods produced their characteristic staining in specimens up to 3 mm thick     

Dichromate-Chlorate Perfusion Prior to Staining Degeneration in Brain and Spinal Cord

A method of fixation compatible with both the Nauta-Gygax and Swank-Davenport procedures for degenerating nerve fibers, which shortens the time required by the former procedure, is as follows: The central nervous system is perfused with a 0.9% aqueous solution of NaCl followed by an aqueous solution containing 5% K₂Cr₂O₇ and 2.5% KClO₃. The central nervous system is then hardened in 10% formalin for 1-3 days. Tissue for Marchi-type staining can be taken at this stage. For silver staining, the processing is continued by immersion overnight in 10% formalin in 20% alcohol, and frozen sections cut the next day. Sections, up to 50μ in thickness, are collected in 10% formalin and impregnated by the Nauta-Gygax technique. Best results are obtained by impregnating within 24-48 hr after sectioning.     

Staining Chromosomes in Sections of Germinal Vesicles of Sarcoptid Mites by a Schiff Reagent

Chromosomes of oocytes, especially early prophase I stages, of Acaridae and Anoetidae species are difficult to stain by procedures using hematoxylin, Feulgen and aceto-orcein. Hematoxylin stains are intensely polychromatic in oocytes; the standard Feulgen procedure is negative with chromosomes during diffuse prophase stages. Satisfactory staining can be obtained with a supersensitive Schiff reagent (Tobie, W. C., Ind. Eng. Chem., Anal. Ed., 14: 405—406, 1942) made by reducing basic fuchsin with gaseous SO₂. Routinely prepared paraffin sections of mites fixed in Carnoy's 6:3:1 mixture were hydrolysed 5-8 min in 1 N HCl, washed well, and stained in this reagent: 1-2 hr for prophase oocytes, 10-20 min for condensed chromosomes. A second staining in a 0.5% aqueous solution of toluidine blue 0, adjusted to pH 5.3-5.5 with a citrate buffer, served to darken the original Feulgen stain. Counterstaining with 0.1-0.2% fast green FCF in the last fluid of the dehydrating series enhanced contrast between chromosomes and cytoplasm. This staining technic is also suitable for preparing whole mounts of mites.     

A Tribasic Stain for Thin Sections of Plastic-Embedded, OsO4-Fixed Tissues

Thin (0.5-1 μ) sections of plastic-embedded, OsO₄-fixed tissues were attached to glass slides by heating to 70 C for 1 min. A saturated solution combining toluidine blue and malachite green was prepared in ethanol (8% of each dye) or water (4% of each dye). Methacrylate or epoxy sections were stained in the ethanol solution for 2-5 min. The water solution was more effective for some epoxy sections (10-80 min). Epoxy sections could be mordanted by 2% KMnO₄, in acetone (1 min) before use of the aqueous dye, reducing staining time to 5-10 min and improving contrast. Aqueous basic fuchsin (4%) was used as the counter-stain in all cases; staining time varied from 1-30 min depending upon the embedding medium and desired effects, methacrylate sections requiring the least time. In the completed stain, nuclei were blue to violet; erythrocytes and mitochondria, green; collagen and elastic tissue, magenta; and much and cartilage, bright cherry red. Sections were coated with an acrylic resin spray and examined or photographed with an oil-immersion lens.     

Controlled Differentiation of Cells and Connective Tissue Fibers in Silver Staining

Controlled silver staining of connective tissue fibers and sometimes of these fibers and cells simultaneously can be obtained. 1. Fix in 10% formalin. Embed in paraffin and cut sections as usual, but do not mount them on slides. Deparaffinize and hydrate through xylene, alcohols and distilled water and henceforth treat them the same as frozen sections. Real frozen sections can also be used. 2. Treat with a freshly prepared 1% solution of KMnO₄, usually 15-60 sec, sometimes up to 10 min. 3. Wash in distilled water, 5-10 sec. 4. Decolorize in 2% potassium metabisulfite, 10-20 sec. 5. Place in distilled water, 1 min. 6. Sensitize with 2% iron alum, 1 min. 7. Place in distilled water, 1 min. 8. Impregnate in Gomori's silver oxide solution, 2 min. 9. Wash in a 1.5% aqueous solution of pyridine, about 15 sec. 10. Reduce in a mixture containing 0.25% gelatin and 2% formalin 1 min. 11. Repeat steps 7 to 10 once or several times until the connective tissue fibers are completely stained. For cell staining (which may fail) proceed as follows: After the first insufficient staining of the connective tissue fibers, rinse in distilled water, dip for 1 sec in Gomori's solution and reduce immediately in gelatin-formalin without previous washing in pyridined water. This step can be repeated. 12. If the staining is too strong, decolorize as needed in 2% iron alum. 13. Toning in 0.2% gold chloride, 5 min or more, followed by fixation in 5% sodium thiosulfate, 1 min, is optional. Counterstain as desired. 14. Wash in tap water, dehydrate, clear in xylene and mount in balsam. The same technique applied to sections attached to slides gives good results but inferior to that obtained in paraffin sections processed in the loose, unmounted condition.     

Block Staining of Nervous Tissue with Silver IV. Embryos

Procedures having enhanced reliability over older methods for both Bielschowsky and Cajal types of stain are described.

Fixation of embryos in a solution containing 4% formaldehyde and 0.5% trichloracetic acid greatly improved the staining of neural elements by Bielschowsky's method.

Among the variations of Cajal's type of staining tried, a modification of Ranson's pyridin-silver method gave the most complete staining of neurofibrillar elements. Washing for 0.5 to 1 hour after silver impregnation and shortening of the reduction time from 24 to 4 hours corrected the tendency of the method to overstain.     

Previsible silver staining of protein in electrophoresis gels with mass spectrometry compatibility

A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1 h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1 h with a detection limit of 0.2 ng/band.     

Oxazine Dyes. I. Celestine Blue B with Iron as a Nuclear Stain

It is suggested that celestine blue B can stain as a colloidal dispersion, the nuclear specificity of which is controlled by the pH. The staining solution is prepared by adding 0.5 ml of concentrated H₂SO₄ to 1 gm of celestine blue B and dissolving the resultant granular mass in 100 ml of 2.5% ferric alum containing 14 ml of glycerol. Sections of amphibian, avian, and mammalian tissue placed for 1 min in this solution and then rinsed in water show as sharp nuclear staining as that usually produced by hematoxylin. A wide variety of fixatives is permissible. Overstaining is not possible within reasonable limits of exposure and no differentiation nor bluing is required. Both the staining solution and stained slides are stable.     

Silver Impregnation of the Golgi Apparatus, with Subsequent Nitrocellulose Embedding

The appearance of silver impregnation of the Golgi apparatus can be enhanced by the use of nitrocellulose as an embedding medium. Fixation of 1.5 mm thick pieces of fresh tissue for 8 hr in: glycine, 1.7 gm; 15% formalin, 100 ml; HNO₃, conc., 0.5 ml, at pH 2.6 followed by rinsing in water, 4 hr in 1.5% AgNO₃, another rinse, and 2 hr reduction in 1.5% hydroquinone in 15% formalin. This staining procedure yields consistently good results for rat, rabbit, and human tissues. Low-viscosity nitrocellulose embedding is done by infiltrating at 56 C in 7% nitrocellulose for 0.5 hr, 15% for 4 hr, and 27% for 1 hr. The nitrocellulose is hardened 2 hr in chloroform, after which, sections as thin as 5 μ can be cut on a sliding microtome. Gold toning and counterstaining can be done with the tissue affixed to the slide. The Golgi apparatus is stained dark brown to black, and there is better preservation of cellular detail than in tissues processed in paraffin.     

Silver staining of DNA synthesizing cells in the neural tube of chick embryos

I report a study by light microscopy of the spinal cord of early chick embryos stained with the ammoniacal silver carbonate solution of Del Rio Hortega. Cell nuclei are stained in a selective fashion and two classes of nuclei - dark and pale - can be distinguished in the neuroepithelium. Neuronal nuclei also show a characteristic staining pattern. A radioautographic study after [3H] thymidine incorporation has shown that it is the dark neuroepithelial nuclei that are engaged in DNA synthesis. Dark nuclei disappear after administration of cytosine arabinoside, supporting the association between DNA synthesis and silver staining of neuroepithelial nuclei.     

Detailed Studies on the Application of Three Fluorescent Antibiotics for Dna Staining in Flow Cytometry

The effects of pH, ionic strength, stain concentration, magnesium concentration, and various fixative agents on DNA staining with the fluorescent antibiotics olivomycin, chromomycin A3, and mithramycin were examined with DNA in solution and in mammalian cells. Ethanol-fixed Chinese hamster cell populations (line CHO) stained with mithramycin and analyzed by flow cytometry provided DNA distribution patterns with a high degree of resolution. Glutaraldehyde-fixed cells exhibited about one-half the fluorescence intensity of ethanol-fixed cells; however, the percentages of cells in G₁, S, and G₂ + M were comparable. DNA distributions obtained for formalin-fixed cells were unacceptable for computer analysis. Cell staining over a pH range of 5-9 in solutions containing 0.15-1 M NaCl and 15-200 mM MgCl₂ provided optimal results based on the DNA profiles obtained by flow cytometry. The intensity of cells stained in 1 M NaCl was one and one-half times greater than cells stained in the absence of NaCl; however, spectrophotofluorometric analysis of mithramycin-magnesium-DNA complexes in solution revealed no significant changes in fluorescence intensity over a range of 0-1.75 M NaCl. These results and those obtained by flow cytometry analysis indicate that the increase in fluorescence of stained cells as a function of increasing ionic strength is due to changes in chromatin structure, providing a larger number of binding sites for the dye-magnesium complex.     

Adaptation of the Nauta Method for Use on Degenerating Fibers in Rat Cortex and Thalamus

Lesions produced in the cerebral cortex of rats were studied by Nauta's method for degeneration. The brains were perfused with physiological NaCl solution, followed by 10% neutral (CaCO₃) formalin. The brains were removed and stored in the formalin for 2 wk to 1 yr. Experimental modifications of the staining method showed that its sensitivity for fine degenerating fibers could he enhanced by the following changes: (a) omitting 0.05% potassium permanganate; (b) replacing the hydroquinone-oxalic acid mixture with 0.1% pyrogallol. Procedure: (1) frozen sections to water; (2) 0.5% phosphomolybdic acid, 45 min; (3) distilled water, 1 min; (4) 0.1% pyrogallol (aq.), 2 min; (5) distilled water, 3 washes of 1 min each; (6) 1.5% silver nitrate (aq.), 30 min; (7) distilled water, 1 min, (8) Laidlaw's ammoniated silver carbonate, 10110 sec; (9) Nauta's reducer, 1-2 min; (10) distilled water, 1 min; (11) 1.0% Na₂S₂O₃, 2 min; (12) distilled water 3 changes, 1 min each; (13) dehydrate, clear, and cover. This method gave equally good results on degenerating axons in both cortex and thalamus.