Fink-Heimer Silver Impregnation of Degenerating Axons and Terminals in Mounted Cryostat Sections of Fresh and Fixed Brains

The silver impregnation method of Fink and Heimer (Brain Res., 4: 369-74, 1967) has been used on cryostat sections of both fresh frozen and formalin-fixed brain tissue mounted on slide. The fixed brains were soaked in 25% sucrose for 2-3 days before freezing. The slides used for mounting were dipped in a 0.5% aqueous gelatin solution containing 50 mg of chrome-alum per 100 ml, drained and allowed to dry in a vertical position. Sections of fresh tissue were fixed for 16 hr in a 10% formalin solution buffered with phosphates to pH 7.0. Staining was carried out according to the Fink-Heimer procedure II and gave results comparable to those obtained on unmounted frozen sections of formalin-fixed material     

Application of the Nauta-Gygax Technic for Degenerating Axons to Mounted Sections

Frozen sections of formalin-fixed brains containing lesions were mounted on slides that had been coated first with albumen-glycerol (1:1) then 4% gelatin and blotted. The slides were placed in formaldehyde vapor at 56° C for 40-60 min, washed, and stored (optional) in 10% formalin-saline. The staining technic was as follows: after washing, soak 30-40 min in 0.5% phosphomolybdic acid, rinse; put in 0.05% potassium permanganate 9-16 min (usually 12 min); decolorize in a 1:1 mixture of 1% hydroquinone and 1% oxalic acid; wash thoroughly; soak in 1.5% AgNO₃ at about 20° C for 25-35 min; rinse; put into an ammino-silver solution (4.5% AgNO₃, 20 ml; pure ethanol, 10 ml; ammonia, sp. gr. 0.880, 2.4 ml; 2.5% NaOH, 1 ml) for 1-2 min; reduce in acidified formalin (distilled water, 400 ml; pure ethanol, 45 ml; 1 % citric acid, 13.5 ml; 10% formalin, 13.5 ml) for 1-3 min; wash; dehydrate through ascending grades of alcohol, including absolute; coat with 0.5% collodion, allow to dry slightly and harden in absolute alcohol-chloroform (2:1); rehydrate and put into 1% Na₂S₂O₃ for 1 min; dehydrate and cover.     

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.     

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.     

Silver Impregnation of Degenerating Axons; Comparisons of Postoperative Intervals, Fixatives and Staining Methods

By introducing variations in procedures and comparing density and patterns of degeneration exhibited, we found that: (1) in the rat, cat, and monkey, the optimum survival time was about 1 wk; this period yielding the maximum amount of argyrophilic reaction and the most consistent staining among several different parts of the brain. Survival times of up to 3 wk did not seriously impair the interpretation of argyrophilial patterns; (2) some qualitative changes in the nuclear patterns of degeneration suggested that the use of a range of survival times may provide useful data; (3) staining differences and similarities in different neural systems of known connections were about the same in the 3 species of animals; (4) formalin, buffered formalin, and a 4%:1% paraformaldehyde-glutaraldehyde mixture were satisfactory perfusion solutions; however, staining was less intense when the Nauta-Gygax method followed the last fixative mixture; (5) The Fink-Heimer I method yielded more consistent results than did the Fink-Heimer II or the Nauta-Gygax methods.     

Silver Impregnation of Peripheral and Central Axons

A silver impregnation method suitable for peripheral and central nervous system axons is described. Essential features are the use of reagent grade chemicals only, a pretreatment solution to ensure optimal impregnation of different organs from different animals and species, and an unvarying procedure. The results are compared to those obtainable with a number of current impregnation methods and with modern immunocytochemical reactions.     

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.     

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.     

A Silver Impregnation Technique for Normal Axons in the Human Central Nervous System for Celloidin and Epon Sections, with Substitutes for Soft Tap Water

An improved silver technique has been developed for human CNS axons in sections from celloidin blocks that resist impregnation because of prolonged storage in alcohol. This method also gives consistently good impregnation of recently fixed material, and thus is suitable for routine use. Slightly modified, the method is also successful with osmicated Epon embedded sections. The quality of silver impregnation in methods using tap water in the reducing solutions varies in different laboratories. Having established that hard water is essential, substitutes for soft water were sought and found.     

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.     

Differential Impregnation of Degenerating Nerve Fibers in Paraffin-Embedded Material

The silver method of Nauta and Gygax (1951) has been used on paraffin embedded material to give a result closely comparable to that obtained by Nauta and Gygax (1954) on frozen sections. It has been found that pyridine plays an important part in suppressing the impregnation of normal fibers in paraffin embedded material and that this sup pression can be augmented by the use of some of the higher methylated derivatives of pyridine, particularly 2,4,6-trimethyl pyridine (collidine).     

Strands in Sections of Sieve Elements Cut in a Cryostat

Observations of these strands are consistent with the idea of trans-cellular strands of cytoplasm in sieve tubes.     

Concomitant Localization of Dopa Oxidase and Adenosine Triphosphatase in Cryostat Sections

Cryostat sections fixed 10 min in calcium formalin were incubated sequentially in 0.1% DOPA, 2 hr at 37 C, and ATPase substrate 1% hr at 37 C. The enzymatically produced calcium phosphate was visualized by 0.2% glyoxal-bis(2-hydroxyanil) (GBHA) in alkaline ethanol as a red precipitate. Nonspecific protein-bound calcium ions which obscured active sites in such formalin-fixed material were successfully removed by treatment with 0.5% ethylenediaminetetraacetic acid at pH 7.0 for 1 min before treatment with GBHA. Phosphatase sites were red; DOPA active sites, black. The method was also successfully applied to the demonstration of alkaline phosphatases. Acetone fixation inhibited both enzymes; fixation in 70% alcohol suppressed the DOPA reaction and partially inhibited ATPase.     

Estimation of Thy-1 in Cryostat Sections of Nervous Tissue

The conventional assay for measuring cell surface antigens--the quantitative absorption of antibody by tissue homogenates--proved inadequate when used to determine the level of Thy-1 glycoprotein in rat nerves and peripheral ganglia. In this paper we report that the binding of 125I-labelled Fab fragments of a monoclonal anti-Thy-1 antibody to cryostat sections is sufficiently sensitive to give consistent estimates of the Thy-1 level on single samples of even small nerves. Observed levels of Thy-1 were generally higher than had previously been thought, and in particular we found no nerves totally lacking the antigen. The lowest levels (6-10 pmol/mg protein) were in peripheral nerves with a large motor component. Autonomic and sensory nerves had higher levels (15-20 pmol/mg protein). The highest levels were on the optic nerve (34 pmol/mg protein), superior cervical sympathetic ganglion (40 pmol/mg protein), and the cerebellar vermis (46 pmoles/mg protein; the only brain region examined in this study). From a practical point of view, the cryostat assay has the advantage that measurements of Thy-1 can be done on sections from the same series as is used for immunohistochemical localisation.     

A Modified Silver Impregnation Technique for the Observation of Thick Sections of Lymph Nodes Perfused with Colloidal Carbon

For many years, a variant of the silver impregnation technique of Bielchowsky has been used to study the lymph node because it clearly outlines the various structures which are usually hard to contrast with standard staining methods. Like other variants of silver impregnation, this method blackens the cell nuclei as well as the reticular fibers; however, it inhibits the impregnation of the nuclear chromatin immediately adjacent to fibers. Hence, this variant selectively darkens the lymphoid cell populations of the nodal structures which contain a loose fiber network.

To study the blood vascular network of the lymph node based on perfusion of colloidal carbon, a staining procedure was needed which would contrast nodal structures on thick sections, while allowing the carbon-filled small blood vessels to be distinguished from the impregnated coarse reticular fibers. In an attempt to adapt this variant of Bielchowsky's technique, 10, 20, 40 and 60 nm thick sections from rat nodes, fixed in a solution of Bouin-Hollande for 72 hr, were silver impregnated with serial dilutions (1:2 to 1:128) of the ammoniacal silver solution. Forty-micrometer thick sections impregnated with a 1:16 dilution of the original silver solution at 37 C and for 30 min provided the best results for the conditions.     

Strands in Sieve Tubes in Longitudinal Cryostat Sections of Cucurbita pepo Stems

Rapidly frozen vascular bundles of Cucurbita pepo were cut longitudinallyin a cryostat and observed in a Nomarski microscope. Clearlydefined transcellular strands were seen traversing the luminaof sieve elements and passing through the sieve pores. Someof these were 5-8 µm wide and gave the impression of compactand comparatively rigid structures, while others, appearingas ribbons about 3 µm in diameter, seemed to have collapsedand lost their contents during preparation. Many of the thickerstrands were seen to consist of a discrete boundary and parallelsubstructural elements approx. 1 µm in diameter. The resultsare consistent with our previous work and support Thaine's theoryof sieve tube translocation based on a transcellular strandsystem.     

Effects of Sunlight on Stained Sections Mounted in Various Media

Sections stained with haematoxylin and eosin showed maximum optical density (OD) at 536 nm, with a second peak at 600 nm. Sections stained with only eosin showed a peak at 536 nm, whilst those stained with haetnatoxylin showed a peak at 600 nm. Reduction in OD at these wavelengths was used to estimate fading of the staining. Direct sunlight reduced the OD of sections mounted in 22 different mounting media by 14 to 64% at 536 nm and 12 to 51% at 600 nm.     

Evidence of Transcellular Strands in Transverse Cryostat Sections of Cucurbita pepo Sieve Tubes

Transverse cryostat sections of rapidly frozen vascular bundlesof Cucurbita pepo were viewed in a microscope with Nomarskioptics. Structural bodies were frequently observed in sievetube lumina and filling the sieve plate pores. The bodies consistof an outer boundary ring and an inner core, often granularin appearance, and may represent transverse sections of boundedtubes filled by substructural material. This evidence is consistentwith earlier observations of strand-like structures with parallelsubstructural elements seen in longitudinal sections.