Silver Staining of Ultrathin Sections of Tissues Embedded in Polyester Resin

Specimens 1 mm³ from rat liver and kidney were fixed for 50 min in cold (0-2° C) 1% OsO₄ in veronal-acetate buffer, pH 7.7, and containing 0.1% MgCl₂; then dehydrated and embedded in Vestopal-W. Sections were cut in two ranges, 0.1-2 µ and 60-90 mµ thick, and attached to slides by floating on water and drying at 60° C. The thicker ones, for light microscopy, were soaked in acetone 1.5-3 hr; the thinner, for electron microscopy, 20-30 min. Both kinds were stained by Wilder's (1935) method for reticulum. Those for light microscopy were finished by dehydrating, clearing and covering in the customary manner; those for electron microscopy, by coating with 1% parlodion, drying, cutting the film about 2 mm² around the section, and freeing the section by soaking in water. The section was then mounted on a grid. The structures stained are: nuclei, basement membrane of capillaries, reticulum fibers of the liver and kidney, and in addition, the basement membrane of the kidney tubules. The mitochondria, vesicles, endoplasmic reticulum and cell membranes were not defined.     

Silver Staining in Clinical Cytogenetics

Silver staining of human chromosomes at prometaphase or metaphase identifies variants in the stalk (nucleolar organizing) regions of acrocentric chromosomes (Nos. 13, 14, 15, 21, 22). Variants are defined by size, number, and morphology of silver staining areas. They are heritable polymorphisms and have not been associated with clinical abnormalities. However, these variants are useful in clinical cytogenetics, specifically in studies attempting to determine 1) whether genetic material has been gained or lost in chromosomal rearrangements, 2) the origin of chromosomal aberrations, 3) the origin of cells in tissue culture, 4) the chromosomal location of single genes, 5) clonal origin of tumors, 6) the zygosity of twins, and 7) paternity. Some chromosomal aberrations require silver staining for their definition. Because loss of the stalk regions per se is apparently not deleterious, demonstration that chromosomal breaks occurred within this region without concomitant loss or gain of genetic material essential for normal human development provides basis for a good prognosis for the individual with the chromosomal rearrangement resulting from such breakage. The principle underlying most of the other applications is to determine whether variants being compared are identical or dissimilar, and to make inferences from these results (e.g., variants in monozygotic twins should all be identical, whereas in dizygotic twins they are as similar as in any pair of sibs). Silver staining is a valuable technique for special questions in clinical analysis.     

Complete Staining of Neuromuscular Innervation with Bromoindigo and Silver

Frozen sections of musde fixed in buffered formaldehyde (pH 7.3) are first incubated for localization of esterase using 5-bromoindoxyl acetate as substrate. The sections are then mounted on slides and stained by a urea-silver nitrate method for axons. Result: subneural apparatus—blue; axons—black; other tissue components in various shades of grey.     

Unfixed Material Embedded in A Methacrylate Resin (Technovit 7100) For Immunofluorescent Staining

Wheat grains were embedded with or without fixation in Technovit 7100 or in paraffin. This enabled us to produce 4 μm sections for imnwnofluorescent staining to see whether serum of patients with baker's asthma contained IgG antibodies against wheat grains. Embedding without fixation in Technovit 7100 appeared to be suitable for unmunofluorescent staining and gave superior results to protocols requiring fixation.     

The Physical Chemistry of Silver Staining

It has been shown that silver deposition plays a part in the silver staining process. From this it has been concluded that the rate of reduction of silver within and on histological structures is an important factor.

Some factors controlling the rate of reduction, such as the adsorption of silver hydroxide and ammonia, the affinity of silver for proteins, and the protective power of the gel structures have been pointed out.

Some simple applications of the ideas to silver staining have been given and two technics described, one making use of piperidine instead of ammonia, the other carrying out the reduction in the presence of the silver solution to facilitate deposition.     

Staining Nerve Fibers with Silver in Tissue Fixed with Bouin's Fluid

Nerve fibers, in organs fixed with Bouin's fluid, are usually refractive to the Davenport silver technic. The axons, however, can be successfully stained if the sections, on slides, are given a preliminary treatment with concentrated pyridine (1 hour), then a 24-hour bath of ammoniated alcohol (99 cc. 80% alcohol, 1 cc 28% ammonium hydroxide) and an interval in 40% aqueous silver nitrate (6-8 hours) before being immersed in the acidified alcoholic silver solution of Davenport. Following the silvering, reduction and toning of the axons, according to the procedure of Davenport, the surrounding non-nervous tissue elements can be counterstained with a combination of either azocarmine, light green and orange G, or azocarmine, aniline blue and orange G.     

Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel

To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.     

A Disrupting Factor in Silver Staining Techniques

Single-stage surface replicas of treated or fresh pollen grains can be made ready for the electron microscope in 1.5 hr. The microspores are discharged into a drop of 50% acetone on a 1 cm square of cleaved mica and air dried. Carbon is evaporated to a film thickness of 35 mμ during rotation of the mica support. The carbon film and microspores are parted from the mica with water and heated in 2-aminoethanol at 145-155 C for 10 min to 3 hr. The replicas are then washed 5 min or longer on water at 90 C and picked up on electron microscope grids. The resulting self-shadowed surface replica can be immediately observed by electron microscopy.     

The Surface Staining of Embedded Tissues

The staining procedure is based on the theory that the freshly cut surface of embedded material will absorb stain only in the exposed tissue elements, provided that the embedding compound itself will not absorb the staining fluid. Concentrated stains are used for short intervals to insure minimum penetration. For paraffin embedded materials: (1) Cut block, preferably on microtome, to the desired tissue surface. (2) Rinse in absolute alcohol. (3) Float face down in stain. (Ripe, concentrated alum hematoxylin—Galigher's formula recommended—will stain in 10 to IS minutes. Heidenhain's iron hematoxylin works exceptionally well in some cases.) Mordant 20% alum 5 to 10 minutes, briefly rinse, and stain comparable 5 to 10 minutes in 1 to 1.5% hematoxylin. (4) Allow to become blue in tap water (for hematoxylin stains). (5) Counter-stain if desired. (6) Dehydrate in absolute alcohol for not more than 10 minutes. (7) Dry for 15 to 20 minutes. (8) Trim block to 2-3 mm. and mount between two cover glasses by use of microflame. Attach mount to slide with balsam. For celloidin embedded materials: (1) Dehydrate block with 90% alcohol, phenol-toluene, finally pure toluene. (2) Rinse cut surface with 90% alcohol, then apply stain. (3) Wash, after hematoxylin stains, counterstain if desired. (4) Dehydrate surface, 90% alcohol, phenol toluene, pure toluene, and mount in medium dissolved in toluene.

Possible applications of surface staining technic are suggested and illustrated.     

Silver Staining of Nerve Fibers in Cardiac Tissue

Fresh hearts of dog were perfused through the coronary vessels with 1000 ml. of fixative (chloral hydrate, 5 g. per 100 ml. of 70% ethyl alcohol) and blocks of tissue 2 × 5 mm. from epicardium to endocardium fixed 48 hours in the same fixative. The blocks were placed in 95% alcohol containing 0.3% addition of strong ammonia for 4 hours, followed by 2 changes of plain 95% alcohol of 1 hour each, then cleared and infiltrated with paraffin. Mounted sections 12-15 µ thick were incubated in 1% silver proteinate (obtained from Serumvertrieb, Marburg, Germany)² at 38° C. for 48 hours in the presence of 10 g. of 15 gauge copper wire per 200 ml. of solution. The slides were rinsed gently in 3 changes of distilled water for 2 minutes, 1 minute and 1 minute, respectively, and reduced in 1% hydroquinone and 5% sodium sulfite for 5 minutes. They were washed 5 minutes in tap water and 5 minutes in 2 changes of distilled water and toned 3-5 minutes in 0.25% gold chloride, rinsed in distilled water 10 seconds, reduced 10 seconds in 1 % oxalic acid, rinsed 1 minute, fixed in 5% sodium thiosulfate 5 minutes, washed in tap water through 3 changes, dehydrated, cleared and covered. All solutions were made with distilled water except where otherwise specified. The results gave good impregnation of fine nerve fibers without the usual confusing staining of reticular tissue.     

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.     

Complete Staining of Nerve Fiber and Myoneural Junctions with Acetylthiocholine and Silver

We describe a combined stain for simultaneous demonstration of the preterminal axons and cholinesterase activity at myoneural junctions of mammalian muscles. This technique employs acetylthiocholine iodide as the substrate for cholinesterase activity and silver nitrate impregnation of preterminal axons. The procedure is rapid, simple and Uses fresh muscles. Intramuscular nerves, preterminal axons and myoneural junctions are stained simultaneously brown or black with minimal background staining of connective tissue and muscle fibers.     

Complete Staining of Nerve Fiber and Myoneural Junctions with Acetylthiocholine and Silver

We describe a combined stain for simultaneous demonstration of the preterminal axons and cholinesterase activity at myoneural junctions of mammalian muscles. This technique employs acetylthiocholine iodide as the substrate for cholinesterase activity and silver nitrate impregnation of preterminal axons. The procedure is rapid, simple and Uses fresh muscles. Intramuscular nerves, preterminal axons and myoneural junctions are stained simultaneously brown or black with minimal background staining of connective tissue and muscle fibers.     

Synthesis of Silver Proteinates for Neurological Staining

Soluble derivatives of the AgNO₃ precipitates of various split protein products were prepared by dissolving the precipitate in a 30-40% aqueous solution of pharmaceutical peptone (Cudahy). The split proteins used included pepsin, trypsin and papain digests of albumin, globulin, gelatin, casein, protamine, and tissue proteins from heart, liver and brain; also, an Escherichia coli digest of casein, and commercial products: Amigen (Mead), casein hydrolysate (Squibb) and pharmaceutical peptone (Cudahy). Staining reactions of the silver derivatives were tested on mammalian nervous tissue. The objective of finding a silver-protein compound that stained axis cylinders selectively was attained only with redissolved silver precipitates of pharmaceutical peptone and bacterially digested casein. It was concluded that the manner of degrading a protein prior to combining it with silver was the most important factor in determining the subsequent staining reaction.     

Hematoxylin Staining of Tissues Embedded in Epoxy Resins

Sections of 0.5-2 μ thickness are affixed to slides with albumen adhesive, thoroughly dried, and placed in xylene or toluene for 1 hr, then brought through ethanol to water. Sections of tissue fixed in O_sO₄ are treated first in 0.1% KMnO₄, then with 1.0% oxalic acid, and after rinsing, incubated at 60 C for 12-24 hr in hematoxylin (Harris's or Ehrlich's) and counterstained 10-15 min with 0.5% phloxine B. Permanent preparations are made by clearing and mounting in a synthetic resin. The method requires only easily available reagents and is suitable for routine processing of epoxy sections.     

A Method for Combined C-Banding and Silver Staining

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.     

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.