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.     

Stabilization of Silver Chromate Golgi Impregnation in Rat Central Nervous System Neurons Using Photographic Developers. I. Light Microscopy

Deterioration of Golgi impregnation begins immediately after impregnated tissue blocks are sectioned with the Vibratome. The first signs of deterioration are fading of delicate impregnated processes, the disruption and fragmentation of dendrites, and, eventually, fading of entire neurons. These changes can be prevented by stabilization, i.e., by converting the water soluble silver chromate Golgi precipitate into metallic silver or by replacing the silver with some other dense, insoluble material. A technique is described using photographic developers to treat Vibratome sections containing Golgi-rapid or Golgi-Kopsch impregnated CNS neurons. In this way part of the silver chromate Golgi precipitate is reduced to metallic silver, and the remaining silver chromate is then removed with sodium thiosulfate. Of the various developers tested, Kodalith and Elon-ascorbic acid gave the best results, with excellent stabilization of the most delicate stuctures, such as the stalks of dendritic spines and finely woven axonal plexuses. Treatment with other developers (HC-110, Neutol, D-19, D-76, D-163, Kodak Universal, Rodinal, Atomal, Diafine, Eukobrom, Microdol-X) resulted in stabilization ranging from good to poor. Good stabilization of Golgi impregnation could also be achieved by first exposing the sections to sodium bromide (bromide substitution) followed by treatment with D-19, Kodalith, Elon-ascorbic acid or HC-110. After stabilization, the sections can be counterstained with aqueous cresyl violet or with alcoholic thionin without degradation of the stabilized Golgi image. The countentain permits exact determination of the position of impregnated neurons in cortical layers or subcortical nuclei.     

Stabilization of silver chromate Golgi impregnation in rat central nervous system neurons using photographic developers. I. Light microscopy

Deterioration of Golgi impregnation begins immediately after impregnated tissue blocks are sectioned with the Vibratome. The first signs of deterioration are fading of delicate impregnated processes, the disruption and fragmentation of dendrites, and, eventually, fading of entire neurons. These changes can be prevented by stabilization, i.e., by converting the water soluble silver chromate Golgi precipitate into metallic silver or by replacing the silver with some other dense, insoluble material. A technique is described using photographic developers to treat Vibratome sections containing Golgi-rapid or Golgi-Kopsch impregnated CNS neurons. In this way part of the silver chromate Golgi precipitate is reduced to metallic silver, and the remaining silver chromate is then removed with sodium thiosulfate. Of the various developers tested, Kodalith and Elon-ascorbic acid gave the best results, with excellent stabilization of the most delicate structures, such as the stalks of dendritic spines and finely woven axonal plexuses. Treatment with other developers (HC-110, Neutol, D-19, D-76, D-163, Kodak Universal, Rodinal, Atomal, Diafine, Eukobrom, Microdol-X) resulted in stabilization ranging from good to poor. Good stabilization of Golgi impregnation could also be achieved by first exposing the sections to sodium bromide (bromide substitution) followed by treatment with D-19, Kodalith, Elon-ascorbic acid or HC-110. After stabilization, the sections can be counterstained with aqueous cresyl violet or with alcoholic thionin without degradation of the stabilized Golgi image. The counterstain permits exact determination of the position of impregnated neurons in cortical layers or subcortical nuclei.     

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.     

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.     

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.     

Stabilization of Silver Chromate Golgi Impregnation in Rat Central Nervous System Neurons Using Photographic Developers. II. Electron Microscopy

The silver chromate precipitate present in neurons impregnated according to the Golgi-rapid and Golgi-Kopsch procedures can be stabilized by treatment with a photographic developer. In a complementary light microscopic study the stabilizing properties of various photographic developers were tested. Kodalith, Elon-ascorbic acid, HC-110, D-19 and Neutol proved to be the most successful. In the present electron microscopic study, we studied the distribution, shape and size of the particles found in Golgi-rapid and Golgi-Kopsch-impregnated neurons by treatment with each of these developers and, simultaneously, the effect of the developer on the preservation of the ultrastructural details. The reaction product after developer-treatment of Golgi-rapid material is sufficiently stable to withstand embedding and thin sectioning, whereas in Golgi-Kopsch material additional gold chloride “Honing” is necessary. In Golgi-impregnated, Kodalith-, Elon-ascorbic acid-, or HC-110-treated material the formed particles are small and located in the cytoplasm, limited by the plasma membranes of the impregnated profiles. In Golgi-impregnated, D-19 treated neurons, the formed particles are relatively coarse. The majority of these particles are within cytoplasm, but particles may also lie either across or entirely outside the plasma membranes of the impregnated profiles. A large number of the small particles in Golgi impregnated, Neutol-stabilized neurons can be seen partly or entirely outside the plasma membranes of the impregnated profiles. Good original ultrastructural preservation seems to be unaffected by developer treatment. Treatment of Golgi material with sodium bromide before stabilization (bromide substitution) results in the formation of small silver particles both inside and outside the impregnated profiles. The sodium bromide step of this procedure has an adverse effect on the preservation of ultrastructural detail.     

The Golgi silver impregnation method: commemorating the centennial of the Nobel Prize in medicine (1906) shared by Camillo Golgi and Santiago Ramón y Cajal

The Golgi silver impregnation technique is a simple histological procedure that reveals complete three-dimensional neuron morphology. This method is based in the formation of opaque intracellular deposits of silver chromate obtained by the reaction between potassium dichromate and silver nitrate (black reaction). Camillo Golgi, its discoverer, and Santiago Ramón y Cajal its main exponent, shared the Nobel Prize of Medicine and Physiology in 1906 for their contribution to the knowledge of the nervous system structure, Their successes were largely due to the application of the silver impregnation method. However, Golgi and Cajal had different views on the structure of nervous tissue. According to the Reticular Theory, defended by Golgi, the nervous system was formed by a network of cells connected via axons within a syncytium. In contrast, Cajal defended the Neuron Doctrine which maintained that the neurons were independent cells. In addition, Golgi had used a variant of his "black reaction" to discover the cellular organelle that became known as the Golgi apparatus. Electron microscopy studies confirmed the postulates of the Neuron Doctrine as well as the existence of the Golgi complex and contributed to a resurgence of use of the Golgi stain. Although modern methods of intracellular staining reveal excellent images of neuron morphology, the Golgi technique is an easier and less expensive method for the study of normal and pathological morphology of neurons.     

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.     

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.     

Stabilization of silver chromate Golgi impregnation in rat central nervous system neurons using photographic developers. II. Electron microscopy

The silver chromate precipitate present in neurons impregnated according to the Golgi-rapid and Golgi-Kopsch procedures can be stabilized by treatment with a photographic developer. In a complementary light microscopic study the stabilizing properties of various photographic developers were tested. Kodalith, Elon-ascorbic acid, HC-110, D-19 and Neutol proved to be the most successful. In the present electron microscopic study, we studied the distribution, shape and size of the particles found in Golgi-rapid and Golgi-Kopsch-impregnated neurons by treatment with each of these developers and, simultaneously, the effect of the developer on the preservation of the ultrastructural details. The reaction product after developer-treatment of Golgi-rapid material is sufficiently stable to withstand embedding and thin sectioning, whereas in Golgi-Kopsch material additional gold chloride "toning" is necessary. In Golgi-impregnated, Kodalith-, Elon-ascorbic acid-, or HC-110-treated material the formed particles are small and located in the cytoplasm, limited by the plasma membranes of the impregnated profiles. In Golgi-impregnated, D-19 treated neurons, the formed particles are relatively coarse. The majority of these particles are within cytoplasm, but particles may also lie either across or entirely outside the plasma membranes of the impregnated profiles. A large number of the small particles in Golgi impregnated, Neutol-stabilized neurons can be seen partly or entirely outside the plasma membranes of the impregnated profiles. Good original ultrastructural preservation seems to be unaffected by developer treatment. Treatment of Golgi material with sodium bromide before stabilization (bromide substitution) results in the formation of small silver particles both inside and outside the impregnated profiles. The sodium bromide step of this procedure has an adverse effect on the preservation of ultrastructural detail.     

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 strains 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 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 rapid method combining Golgi and Nissl staining to study neuronal morphology and cytoarchitecture.

The Golgi silver impregnation technique gives detailed information on neuronal morphology of the few neurons it labels, whereas the majority remain unstained. In contrast, the Nissl staining technique allows for consistent labeling of the whole neuronal population but gives very limited information on neuronal morphology. Most studies characterizing neuronal cell types in the context of their distribution within the tissue slice tend to use the Golgi silver impregnation technique for neuronal morphology followed by deimpregnation as a prerequisite for showing that neuron's histological location by subsequent Nissl staining. Here, we describe a rapid method combining Golgi silver impregnation with cresyl violet staining that provides a useful and simple approach to combining cellular morphology with cytoarchitecture without the need for deimpregnating the tissue. Our method allowed us to identify neurons of the facial nucleus and the supratrigeminal nucleus, as well as assessing cellular distribution within layers of the dorsal cochlear nucleus. With this method, we also have been able to directly compare morphological characteristics of neuronal somata at the dorsal cochlear nucleus when labeled with cresyl violet with those obtained with the Golgi method, and we found that cresyl violet-labeled cell bodies appear smaller at high cellular densities. Our observation suggests that cresyl violet staining is inadequate to quantify differences in soma sizes.     

Mapping of experimental axon degeneration by electron microscopy of golgi preparations

Summary For the mapping of the terminal area of transected axons within the central nervous system, electron microscopy has recently been adopted. A greater accuracy is thereby obtained than with silver impregnation and light microscopy, since it becomes possible to determine the kinds of structure (soma, dendrites, spines) with which the degenerating boutons establish synaptic contact. In the present study this technique was extended by Golgi impregnation of such material with the aim of making possible classification of the postsynaptic neuron. A few days after transection of a pathway (commissural fibres to the hippocampus being used as a model in this study) the brain was fixed by perfusion with phosphate buffered formalin with sucrose. This was followed by immersion in an osmium tetroxide-potassium dichromate mixture (Dalton's fixative without sodium chloride) later replaced by a solution of silver nitrate. Satisfactory Golgi impregnation of nerve cells and processes was obtained. By careful trimming, and reembedding of selected areas, blocks for ultramicrotomy could be obtained which contained only one type of impregnated cell, e.g., hippocampal pyramidal cells.The relation of basal dendrites of such cells to degenerating boutons of commissural fibres was studied. Numerous examples of contact between degenerating boutons and spines belonging to the basal dendrites were seen. Although the Golgi precipitate obscured the postsynaptic substance in the spines, a number of features at the sites of contact were considered strong indication that many of the contacts were synapses and not merely the result of random juxtaposition. This combined procedure is supposed to be applicable to other problems and to other parts of the nervous system as well.This study was supported in part by Grant NB 02215 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. This ais is gratefully acknowledged. The author is indebted to Mrs. J. L. Vaaland and Mr. B. V. Johansen for valuable technical assistance.     

STUDIES ON THE GOLGI APPARATUS BY ELECTRON MICROSCOPY WITH PARTICULAR REFERENCE TO AOYAMA'S TECHNIQUE

1. Aoyama's silver impregnation method for the Golgi apparatus has been used on exocrine cells of the pancreas of the mouse and studied by electron microscopy in order to determine as precisely as possible where the silver is deposited. Similar cells have also been fixed in buffered osmium tetroxide solution and compared with cells treated by the silver technique. 2. Examination of the Aoyama preparations usually revealed a light deposition of silver in the cytoplasm (hyaloplasm or matrix) and a heavy deposition of silver around a series of closely apposed vacuoles. The heavy deposition of silver was regarded as revealing the chromophilic region of the Golgi apparatus while the vacuoles were identified as the chromophobic component. 3. Comparison of the silver preparations with those fixed in buffered osmium tetroxide solution showed that the silver was primarily deposited in the region of the Golgi membranes.     

Studies on the Golgi apparatus by electron microscopy with particular reference to Aoyama's technique

1. Aoyama's silver impregnation method for the Golgi apparatus has been used on exocrine cells of the pancreas of the mouse and studied by electron microscopy in order to determine as precisely as possible where the silver is deposited. Similar cells have also been fixed in buffered osmium tetroxide solution and compared with cells treated by the silver technique. 2. Examination of the Aoyama preparations usually revealed a light deposition of silver in the cytoplasm (hyaloplasm or matrix) and a heavy deposition of silver around a series of closely apposed vacuoles. The heavy deposition of silver was regarded as revealing the chromophilic region of the Golgi apparatus while the vacuoles were identified as the chromophobic component. 3. Comparison of the silver preparations with those fixed in buffered osmium tetroxide solution showed that the silver was primarily deposited in the region of the Golgi membranes.     

Microwaves Applied to Silver Impregnations with Ammoniacal Silver Carbonate

Techniques for impregnation with ammoniacal silver carbonate provide valuable information on all types of tissue; however, the time investment required to impregnate a few sections has limited their application. We have shortened the impregnation times by using microwaves in techniques for reticular fibers, astrocytes, nerve fibers and chromaffin cells. The results were satisfactory with markedly reduced impregnation time and elimination of nonspecific silver deposits.     

The classic Golgi apparatus and vacuoles

The dense vacuoles, considered to be the classic Golgi apparatus in the root meristem ofFagopyrum, were studied by the following methods: 1. Impregnation methods for the demonstration of the Golgi apparatus, 2. cytochemical methods, 3. electron microscopic methods in the light microscope and 4. the electron microscope. A comparison was made with the classic Golgi apparatus in animal cells in the light and electron microscope. Dense vacuoles inFagopyrum and also evidently in other plants, were taken for the classic Golgi apparatus on account of their morphological similarity to the Golgi apparatus in animal cells on impregnation with silver and osmium and their staining preperties with lipoid methods. Dense vacuoles differ from the classic Golgi apparatus in other chemical properties, such as content of phenol substances, etc. No formations were found in animal cells which were similar to dense vacuoles on investigating by electron microscopy. In the electron microscope dense vacuoles have the appearance of derivatives of the normal light vacuoles known in plant cells. They therefore belong to vacuome of plant cell and cannot be analogous to the classic Golgi apparatus in animal cells. Thus the use of the term Golgi apparatus for dense vacuoles is not well founded. A comparison was made of fixation and impregnation used in the light microscope with fixation in the electron microscope. After fixation with permanganate, dense vacuoles have the same shape as after impregnation. After fixation with permanganate, they stain an intense black in the same way as after impregnation with silver and osmium. The form of the vacuoles is dependent on the fixation used. The comparison was made in the light microscope.