Modified Silver Impregnation as a Method for Fish Nervous Tissue Visualization

Journal of Ichthyology - The fishes’ nervous system is one of the key objects of evolutionary and biomedical researches. The multiple approaches and methods for visualizing the nervous tissue...     

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 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.     

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.     

A Silver Impregnation Technique for the Study of Steroid Receptors in Central Nervous System Tissue Prepared for Autoradiography

The commonly used silver stains were found to be unsatisfactory for nervous tissue processed for autoradiography. A silver impregnation procedure for central nervous system tissues prepared for the autoradiographic study of steroid receptors is described. The procedure is a combination of several silver and reticular stains made up in solutions containing dimethylsulfoxide. The technique clearly distinguishes perikarya of neurons, brain nuclei and fiber tracts without substantial loss of silver grains, and thus greatly facilitates the identification of steroid receptor nuclei at all levels of the central nervous system.     

A Method for Silver Impregnation of Mammary Myoepithelia

Histochemical methods for microscopic visualization of nummary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

Silver Impregnation of Ciliated Protozoa by the Chatton-Lwoff Technic

The ciliates are manipulated at all stages by means of micropipettes and fine needles, proceeding by the following steps: Fix the material in a small receptacle with Champy's fluid 1-3 minutes following with Da Fano's solution for several hours. Transfer the specimens to a slide, withdraw excess fluid and embed them in warm (35°-45°C.) gelatin containing 0.05% sodium chloride. Refrigerate in a moist chamber until the gelatin has set, and then immerse 10-20 minutes in 3% silver nitrate (aqueous) at 5-10°C. Wash with cold distilled water, submerge the preparation in cold water to a depth of several centimeters and expose to a strong light for 10-30 minutes. Silver is deposited on various pellicular structures which then appear black in the dehydrated and mounted specimens. Neatly revealed are the many longitudinal and transverse fibrils of the “silverline system”, basal granules of the cilia, bases of buccal ciliary organelles, contractile vacuole pores and the cytoproct. None of these structures, which today are considered to be of inestimable value in comparative morphological and taxonomical studies of ciliates in general, is so precisely made evident by any other technic known to the author.     

Notes on Technic: Mahon's Myelin Stain for Celloidin-Embedded Nervous Tissue

Two methods commonly used to stain myelin sheaths are Kluver and Barrera's luxol fast blue (Kluver and Barrera 1953) and Weil's iron hematoxylin (Weil 1928). Both require differentiation of the stain; in addition, the Kluver-Barrera method specifies 16-24 hour staining. A third method for the selective staining of myelinated axons is that of Mahon (1938), which was introduced for use with paraffin-embedded autopsy tissue. The procedure possesses two distinct advantages since it requires: (1) no differentiation of the stain and (2) only 1 hour staining. Loyez's (1910) myelin stain for celloidin embedded tissue is similar to Mahon's but calls for long staining followed by differentiation. This report describes the application of Mahon's method to celloidin-embedded experimental tissue and emphasizes its utility for staining tissues to be used for reconstructing microelectrode penetrations (fig. 1) and for demonstrating the effect of experimental lesions (fig. 2).     

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 Degenerating Axon Terminals in the Central Nervous System: (1) Technic. (2) Chemical Notes

A new silver technic, tested on the brain of the rat, is described, especially suitable for demonstrating terminal degeneration within the central nervous system. It is a modification of the Glees method, designed to avoid use of tap water in preparing solutions. Some of the chemical principles underlying the process of reductive liberation of metallic silver from ammoniacal silver nitrate solutions are discussed.     

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 μm) sections but normal (10%) for thicker (20 μm) 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 New Method of Silver Impregnation for Nerve Cells and Fibers

Fragments of tissue, immediately after death, are fixed in Debaisieux's modification of the Duboscq-Brazil picro-aceticformol fluid, and treated as follows: Hydrate by soaking 2-6 hr. in distilled water with 30 drops of cone. NH₄OH per 100 cc. Freeze and cut sections about 25μ in thickness. Bleach sections about 15 min. in ammoniacal water (52 drops cone. NH₄OH per 100 cc. water). Transfer to 20% AgNO₃ solution and heat at 45° C. till light brown. Add cone. NH₄OH drop by drop till the Ag precipitates and then redisolves into an opalescent solution. Pour solution and sections into a little distilled water and transfer sections quickly to formaldehyde solution (3 cc. formalin to 100 cc. water). Dip sections in distilled water and transfer to 1% aqueous gold chloride till deep blue. Place for about 10 minutes in 5% aqueous sodium thiosulfate solution for fixing and clearing. Wash thoroly in tap water, dehydrate and mount. Special directions are given for applying this technic to delicate material such as insects, and for use with serial sections.     

A Golgi-Electron Microscope Method for Insect Nervous Tissue

Golgi's light microscopic method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 21/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50μm sections am made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

A Controlled Silver Impregnation Method to Characterize Cultured Cardiomyocytes

A morphological characterization of cultured cardiomyocytes was attempted using a modification of a silver impregnation technique originally described for connective tissue. Cardiac cells, obtained from newborn rats and grown as dissociated cultures on plastic surfaces, were fixed in methanol plus 5% glacial acetic acid, treated with potassium permanganate, decolorized in oxalic acid, sensitized with potassium bichromate, impregnated with a silver-ammonium complex, reduced in gelatin-formalin preparation, toned with gold chloride and fixed in sodium thiosulfate. The cultured cardiac cells tended to form a monolayer, although many myocytes remained isolated. Spherical nuclei, sharply stained with silver, were centrally located and surrounded by relatively plentiful cytoplasm packed with well delineated myofibrils. Contaminating fibroblasts were readily distinguished by their spindle-shaped nuclei and the presence of overstained collagen fibers, as well as the absence of myofibrils. In the absence of specific antibody for immunocytochemical identification of cardiomyocytes, morphological characterization of cell type and degree of differentiation by the controlled silver impregnation procedure described here provides a viable alternative, both in short- and long-term studies.     

An Ammoniated Silver Carbonate Technic for Nervous Structures in Mounted Paraffin Sections

Specimens of both vertebrate and invertebrate nerve-containing tissues were fixed 2-3 days in Bouin's fluid, soaked 2 days in alcohol containing 2% strong ammonia water, dehydrated and embedded in paraffin. The sections were mounted with gelatin adhesive according to Masson's procedure, dewaxed, passed through graded alcohols to water, then back to 2% ammoniated 80% alcohol for 12-24 hours. The slides were rinsed 3-5 seconds in distilled water, impregnated about one and a half hours in 40% AgNO₃ at increasing temperature up to 45°C. The slides were flooded with 62.5% formalin and this solution allowed to remain 3-5 minutes; they were then blotted with filter paper. A second impregnation in ammoniated silver carbonate, controlled under the microscope, was followed by a 10-minute treatment with 10% aqueous acetic acid, toning with gold chloride, then thiosulfate and finally washing. Counterstaining with ponceau red or acid fuchsin, eventually followed by aniline blue or fast green, dehydration and covering, completed the process.     

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.     

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.     

Graded Impregnation of Nervous Tissue Stained by the Golgi Procedure

A simple technique is described by which retinal whole mounts or slices of brain can be impregnated by the Golgi procedure in a graded manner. The unfixed tissue is laid on a glass slide and covered with a layer of Telfa gauze which is tied in place. Following fixation, the progress of staining, which begins beneath holes in the gauze, is followed by direct observation through the slide. The tissue is impregnated or reimpregnated until the desired results are obtained.