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.     

A New Method for Easy Demonstration of Argyrophil Cells

A new method for silver impregnation of endocrine cells of the gastrointestinal mucosa is described. It offers great reliability, eveness of impregnation, and, since it can be used on batches of slides, is also suitable for histology class and investigation material. The procedure for paraffin sections of formalin-fixed material is as follows: dewax and transfer to distilled water, leave in 0.5% silver nitrate solution for 2 hours at 60 C. Rinse in distilled water, then treat in Bodian developer (hydroquinone, 1 g; sodium sulphite, 5 g; distilled water, 100 ml) previously heated to 60 C. Rinse in running tap water, distilled water, and then re-impregnate for 10 minutes at 60 C in the same silver solution and reduoc in Bodian's solution. Sma the background is not impregnated by this method, sections may be counterstained by any basic anilin dye to bring out nuclei. A 0.1% kernechtrot solution was found very satisfactory in this respect. The granulations of argyrophil cells stand out sharply black against a red background.     

A new method for easy demonstration of argyrophil cells.

A new method for silver impregnation of endocrine cells of the gastrointestinal mucosa is described. It offers great reliability, eveness of impregnation, and, since it can be used on batches of slides, is also suitable for histology class and investigation material. The procedure for paraffin sections of formalin-fixed material is as follows: dewax and transfer to distilled water, leave in 0.5% silver nitrate solution for 2 hours at 60 C. Rinse in distilled water, then treat in Bodian developer (hydroquinone, 1 g; sodium sulphite, 5 g; distilled water, 100 ml) previously heated to 60 C. Rinse in running tap water, distilled water, and then re-impregnate for 10 minutes at 60 C in the same silver solution and reduce in Bodian's solution. Since the background is not impregnated by this method, sections may be counterstained by any basic anilin dye to bring out nuclei. A 0.1% kernechtrot solution was found very satisfactory in this respect. The granulations of argyrophil cells stand out sharply black against a red background.     

Suppression of connective tissue impregnation in a silver technique for demonstrating nerve fibers.

A tissue pretreatment is introduced which effectively suppresses the silver impregnation of connective tissue and nonspecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcohol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginning with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilizing fresh frozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation is suppressed by the use of mercuric chloride in the fixative and that the background suppression is related to the short fixation time with formolsublimate.     

Suppression of Connective Tissue Impregnation in a Silver Technique for Demonstrating Nerve Fibers

A tissue pretreatment technique is introduced which effectively suppresses the silver impregnation of connective tissue and nompecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcobol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginnins with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilidng fresh hrozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation H suppressed by the use of mercuric chloride in the fixative and that the background supprgsion is related to the short fixation time with formol-sublimate.     

Histological Technic for Cerebellar Climbing and Mossy Fibers

In order to, avoid disadvantages attendant upon the use of fresh frozen sections, or of block impregnation with silver, in staining climbing or mossy fibers of the cerebellum, Rio Hortega's double impregnation method for nerve fibers is useful. This consists of prolonged formalin fixation prior to cutting frozen sections (which thereafter are easier to cut) and preliminary treatment with ammoniacal aqueous and alcoholic washes, mordanting in pyridine silver, and treatment with pyridine-silver-carbonate. Following this, sections are handled individually through one of several reduction methods after which they may be directly mounted or gold toned.     

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.     

A method for silver impregnation of mammary myoepithelia

Histochemical methods for microscopic visualization of mammary 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.     

A Controllable Silver Stain for Nerve Fibers and Nerve Endings

A paraffin section method is described with a yellow-brown-black color range comparable to that of Ranson's pyridine silver block stain. After impregnation with activated protargol and reduction with a fine grain photographic developer, silver nitrate impregnation and reduction are repeated as often as necessary. The procedure is as follows:

Place hydrated sections of tissue fixed in chloral hydrate (25 g. in 100 ml. of 50% alcohol) in 1% aqueous protargol (Winthrop Chemical Co.) containing 5-6 g. metallic copper for 12-24 hours. After rinsing in 2 changes of distilled water, reduce 5 to 10 minutes in: Elon (Eastman Kodak Co.) 0.2 g., Na₂SO₃, dessicated, 10 g., hydroquinone 0.5 g., sodium borate powder 0.1 g., distilled water 100 ml. Wash thoroly in 4 or 5 changes of distilled water and place in 1% aqueous AgNO₃ for 10-20 minutes at 28°-50° C. Rinse in 2 or 3 changes of distilled water and reduce in the elon-hydroquinone solution. After thoroly washing in 4 or 5 changes of distilled water, examine under microscope.

If too pale, treat again in silver nitrate for 10-20 minutes, rinse, reduce 5-10 minutes and wash thoroly until nerve fibers show distinct microscopic differentiation, then dehydrate, clear and mount.     

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.     

Chemical Stabilization of Golgi Silver Chromate Impregnations

Blocks of neural tissue were processed by a modified Golgi-Kopsch procedure and by the rapid Golgi method. Following the impregnation, the blocks were embedded in celloidin, sectioned at 100μm, and collected in 70% alcohol. The sections were then processed as follows: 1) rinsed in distilled water; 2) substituted with 0.4M sodium bromide for five minutes; 3) reduced in Kodak D-19 developer; and 4) treated in 0.5M sodium thiosulfate. The silver chromate deposits within the impregnated cells are converted successively to silver bromide and to reduced silver by this procedure. Sections so treated resist decomposition of the Golgi impregnation, and they may be counterstained with conventional aqueous cresyl violet to demonstrate the cytoarchitecture of the Golgi-impregnated tissue.     

Chemical stabilization of Golgi silver chromate impregnations.

Blocks of neural tissue were processed by a modified Golgi-Kopsch procedure and by the rapid Golgi method. Following the impregnation, the blocks were embedded in celloidin, sectioned at 100 micrometer, and collected in 70% alcohol. The sections were then processed as follows: 1) rinsed in distilled water; 2) substituted with 0.4M sodium bromide for five minutes; 3) reduced in Kodak D-19 developer; and 4) treated in 0.5M sodium thiosulfate. The silver chromate deposits within the impregnated cells are converted successively to silver bromide and to reduced silver by this procedure. Sections so treated resist decomposition of the Golgi impregnation, and they may be counterstained with conventional aqueous cresyl violet to demonstrate the cytoarchitecture of the Golgi-impregnated tissue.     

A silver carbonate method for cell counts of neurons and glial elements on paraffin embedded brain tissue.

A modification of the Del Rio-Hortega method for the demonstration of central nervous system elements is presented. This silver impregnation technique is particularly useful for the classification of cell types for quantitative differential cell counts. Formalin fixed paraffin sections are immersed in formol-ammonium bromide for 1 1/2 hours; this solution is an excellent mordant for various silver nitrate stains. The samples are stained for 20 to 60 minutes in a silver carbonate solution (25 ml of 25% silver nitrate combined with 200 ml of 5% sodium carbonate) and then reduced in a 1% formaldehyde solution to which 20 drops of acetic acid have been added. Finally, the slides are fixed in sodium thiosulfate, rinsed in tap water, dehydrated, cleared, and mounted. This procedure will enable this investigator to identify neurons, oligodendroglia, and astrocytes on the basis of their nuclear staining as well as to demonstrate the laminae of brain tissue since the method allows differentiation of cell layers and fiber tracts.     

Modification of the Silver Proteinate Impregnation Technique for Protozoa and Cultured Nerve Cells

Silver impregnation with silver-protein compounds is widely used for staining tissue sections and cell cultures. Some authors report that the results obtained with these methods have not always been reproducible because the reagent's composition varies according to the manufacturer. To avoid this problem in the method described in this paper, a silver proteinate, produced in our own laboratory is used. Although our method is based on Bodian's, the modifications we have made allows its use for both free-living cells (protozoa) and cells grown in culture (nerve cells). The significant modifications are 1) different fixation, 2) postfixation with Cajal's for-mol-bromide, 3) changes in the duration of the impregnation steps technique and 4) elimination of metallic copper. The method reported here enables us to use silver proteinate whenever we require it and to control the composition of the silver proteinate. This technique can be used for cells cultured in either plastic or glass.     

Effects of Oxidation on Neurofibrillar Argyrophilia

The effect of oxidation on neurofibrillar argyrophilia was studied by subjecting nervous tissues containing both normal and degenerating fibers to the action of potassium permanganate, periodic acid, chromic acid, lead tetraacetate, and sodium bismuthate prior to silver impregnation. The argyrophilic response of normal fibers to such treatment was studied with the Nonidez silver nitrate block technic, the double impregnation method of Bielschowsky on both blocks and sections, and a silver proteinate procedure. The response of degenerating fibers was studied by the Cajal formula 6 block technic and the modified Bielschowsky procedure of Nauta and Ryan for sections. The experimental data indicated that such oxidation did not produce any differential staining effects between normal or degenerating fibers.     

Problems and experience in the light optical and histological presentation of glia cells]

1. 8 histological techniques and 13 modifications derived from those were tested on usefulness for the demonstration of glial cells in the adult rat brain. From these methods the impregnation techniques of Golgi-Kopsch, Valenzuela y Chacón and Rio del Hortega were modified according to a scheme of variance to find out the optimal variants. 2. The impregnation quality depends on the animal species, the animal age, the health of brains, the brain area, the balanced proportion of the treatment stages and the biochemical state of the glial cells. 3. The silver impregnation techniques are not so specific that only one glial type is stained, but one type prevails. The silver carbonate procedure according to Hortega allows to impregnate oligodendrocytes, microglial cells and astrocytes in frozen as well as in paraffin sections. The method of Golgi-Kopsch is more suited for oligodendrocytes and microglial cells than for astrocytes. Following the procedure of Valenzuela y Chacón especially astrocytes, but also microglial cells allow impregnation in both frozen and paraffin sections. 4. The different demonstration qualities of the proved methods call for critical examination of absolute measurements of cell size, length of processes and ramification density. 5. The presence of cell groups of different disposition towards impregnation within a glial type speaks for a biochemical inhomogeneity of the glial types.     

Effect of temperature on argyrophil impregnation: development of a high temperature rapid argyrophil procedure

Our studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

Effect of Temperature on Argyrophil Impregnation: Development of A High Temperature Rapid Argyrophil Procedure

Out studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

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 Golgi-electron microscope method for insect nervous tissue.

Golgi's light microscope 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 2 1/2% 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, 50 mum sections are 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.