Evolution of the silver and gold stains in neurohistology

Scientific investigations depend on the reliability of the observations that can be made. This reliability is determined in part by the understanding of the techniques and technology used to make the observations. The limitations and the strengths of the methodology and the equipment used must be evaluated thoroughly. The extent to which this is and has been the case for the use of the metal based stains in neuroscience is the subject of this paper. I evaluate the metallic stains used for neuroscience from several perspectives. I review briefly the state of neurohistology prior to its “golden years,” 1870-1910. Then I trace the development of the silver based stains used for neurohistology. I wanted to discuss the reasoning used by the originators of the silver based techniques in developing their specific procedures, but discovered that while procedures may be published, the methods and ideas used to arrive at the final procedures are not usually described in published work.     

Evolution of the silver and gold stains in neurohistology.

Scientific investigations depend on the reliability of the observations that can be made. This reliability is determined in part by the understanding of the techniques and technology used to make the observations. The limitations and the strengths of the methodology and the equipment used must be evaluated thoroughly. The extent to which this is and has been the case for the use of the metal based stains in neuroscience is the subject of this paper. I evaluate the metallic stains used for neuroscience from several perspectives. I review briefly the state of neurohistology prior to its "golden years," 1870-1910. Then I trace the development of the silver based stains used for neurohistology. I wanted to discuss the reasoning used by the originators of the silver based techniques in developing their specific procedures, but discovered that while procedures may be published, the methods and ideas used to arrive at the final procedures are not usually described in published work.     

Fungal melanin detection by the use of copper sulfide-silver

Silver-staining procedures were investigated for their effectiveness in identifying cell wall-based fungal melanins in live and fixed plastic embedded samples, particularly 1,8-dihydroxynaphthalene (DHN) based polyketide melanins. We developed a simple and reliable melanin-staining technique based on a silver accumulation method originally published for histological demonstration of heavy metal sulfides in mammalian tissues. Copper is bound to fungal melanin followed by formation of the copper sulfide at melanin sites in fungal cell walls, which then are amplified into vivid black stains using a silver enhancement step. The method demonstrates patterns of melanization in a range of fungal hyphae and is suitable for light and electron microscopy. Albino mutant fungi and normally nonmelanized fungi do not stain with the sulfide-silver technique. Mammalian melanocytes also were labeled by the technique, indicating its universality as a melanin probe.     

Silver stains demonstrating neuroendocrine cells

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.     

Silver stains demonstrating neuroendocrine cells

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.     

Model system studies of staining procedures for lysine and arginine residues.

Using polyacrylamide films containg poly-lysine, polyarginine and DNA as test models, a variety of reportedly specific staining procedures have been examine. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin arginine will stain with fast green, if proteins containing both amino-acids are stained with dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acidarginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrocholoric acid in a Feulgen-like procedure which stains DNA to the same level as the classiclal hydrochloric acid based procedure while also staining arginine present.     

A rapid sensitive silver stain for polypeptides in polyacrylamide gels

The use of silver to detect polypeptides was originally achieved by modifying tissue stains. By adapting methods of photochemistry we have developed a new silver stain for polypeptides which is nearly as sensitive but much more efficient than these earlier procedures. The new silver stain utilizes only three solutions and allows protein patterns to be visualized within 50 min. Its sensitivity is 100 times that of the Coomassie blue stain.     

Identification of Pneumocystis carinii in immunodeficient mice

Various procedures were utilized to determine the most sensitive, cost and labor effective techniques for detection of Pneumocystis carinii in immunologically compromised mice. Immunoperoxidase staining techniques that utilized polyclonal antibodies directed against purified rat or mouse P. carinii were more sensitive and specific than staining with Gomori's methenamine silver. Indirect immunofluorescence microscopy on frozen sections was comparable to immunoperoxidase staining, but lacked fine cytologic detail. Impression smears were of limited value when stained with Diff-Quik Stain, Harleco's Hemacolor, Wright-Giemsa or Wright-Leishman stains. However, cysts could be detected consistently in imprints stained with Gomori's methanamine silver. Transmission electron microscopy showed ultrastructural detail of P. carinii, but this technique was too costly and time consuming for routine use. Thus, because of its sensitivity and specificity, immunohistochemistry on paraffin sections was the most satisfactory method for screening and identifying P. carinii in lungs of immunocompromised mice.     

Modified protocols for rapid carrot genomic DNA extraction and AFLP™ analysis using silver stain or radioisotopes

AFLP^™ is one of the most frequently used techniques for identification of molecular markers. We have modified the procedures for genomic DNA extraction, AFLP product generation and silver staining in order to speed up analyses and screen large numbers of plant samples. Using this protocol, we were able to achieve an 82% reduction of costs without compromising the reliability and quality of data gathered.     

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.     

Strategies to improve the reliability of a theory: the experiment of bacterial invasion into cultured epithelial cells

An analysis is presented of published methods that have been used by experimenters to justify the reliability of the theory of invasion of microorganisms into cultured cells. The results show that, to demonstrate this invasion, many experimenters used two or more methods that were based on independent technical and theoretical principles, and by doing so improved the reliability of the theory. Subsequently I compare this strategy of ‘multiple derivability’ with other strategies, discussed in the literature in relation to the mesosome, a bacterial organelle that had been detected with the electron microsope, but which appeared later to be an artifact. I propose that different strategies have been applied in this problem, and multiple derivability may have been the decisive one. Finally I discuss the idea that multiple derivability may help to anchor theories in a larger network of theories.     

A simple procedure to produce monospecific polyclonal antibodies of high affinity against actin from muscular sources

A simple and effective technique to produce monospecific polyclonal antibodies of high affinity against actin is described. In this procedure, rabbit skeletal muscle actin in the 1:1 complex with bovine pancreatic deoxyribonuclease I is used as antigen to immunize rabbits. The antisera obtained are shown to contain antibodies against both actin and deoxyribonuclease I. By affinity chromatography the two antibody preparations were separated and characterized. The affinity-purified anti-deoxyribonuclease I and anti-actin do not show cross-reactivity. Thus, anti-deoxyribonuclease I inhibits the enzymic activity of deoxyribonuclease I and stains the enzyme after Western blotting. Affinity-purified anti-actin does not inhibit deoxyribonuclease I activity and stains only actin after Western blotting. The affinity-purified anti-actin can be used in a number of different actin-detecting techniques such as in immunohistochemistry and in immunoblotting techniques. This antibody recognizes only actins from muscular tissues with high affinity. Immunoblots of polyacrylamide gels in the presence of ampholytes (IEF) indicate that this antibody only recognizes the alpha-variants of actin. Thus, the skeletal and cardiac alpha-actins are recognized but not the smooth muscle gamma-isoform and the cytoplasmic actins. Vascular smooth muscle alpha-actin is not recognized when using immunoblotting or enzyme-linked immunosorbent techniques. On frozen sections, however, the anti-actin antibody clearly stained vascular smooth muscle cells. Epitope analysis using actin fragments generated by limited proteolysis and selective cleavage using hydroxylamine indicate that this antibody is directed against a rather limited region within the N-terminus of actin.     

Differential Staining of Inner Ear Fluids by Protargol

Present day techniques for processing temporal bones involve celloidin embedding. With a few modifications in Bodian's silver staining procedure the celloidin of the endolymphatic spaces stains darker than that of the perilymphatic spaces providing there is no break in the anatomical barrier between them. Essentially the routine procedure of Bodian is used except that metallic copper is omitted from the staining solution, impregnation time is reduced to 3 hr, reduction time is extended to 10 min and no oxalic acid is used for gold toning.     

Silver impregnation methods for reticulum fibers and reticulin: A re-investigation of their origins and specifity

Summary Maresch (1905) introduced Bielschowsky's silver impregnation technic for neurofibrils as a stain for reticulum fibers, but emphasized the nonspecifity of such procedures. This lack of specifity has been confirmed repeatedly. Yet, since the 1920's the definition of reticulin and studies of its distribution were based solely on silver impregnation technics. The chemical mechanism and specifity of this group of stains is obscure. Application of Gomori's and Wilder's methods to human tissues showed variations of staining patterns with the fixatives and technics employed. Besides reticulum fibers, various other tissue structures, e.g. I bands of striated muscle, fibers in nervous tissues, and model substances, e.g. polysaccharides, egg white, gliadin, were also stained. Deposition of silver compounds on reticulum fibers was limited to an easily removable substance; the remaining collagen component did not bind silver. These histochemical studies indicate that silver impregnation technics for reticulum fibers have no chemical significance and cannot be considered as histochemical technics for reticulin or type III collagen.     

The staining of elastic fibres with Direct Blue 152. A general hypothesis for the staining of elastic fibres

Summary Elastic fibres may be stained by a number of dyes, e.g. Direct Blue 1 (C.I. 24410), Direct Blue 10 (C.I. 24340), Direct Blue 15 (C.I. 24400), Direct Blue 152 (C.I. 24366) and Direct Violet 37 (C.I. 24370). A convenient method using Direct Blue 152 has been developed which is specific for elastic fibres. The method is simple and allows the demonstration of other connective tissue fibres. Staining of elastic fibres is unimpaired by numerous blocking procedures or by changes in dyebath pH. These properties are shared by several standard elastic fibre stains.As the Direct dyes and several of the standard elastic fibre stains possess numerous aromatic rings a wide range of dyes containing varying numbers of aromatic rings were examined for ability to stain elastic fibres. No association was observed between the ability to stain elastic fibres and dye class, formal charge or the presence of hydrogen bonding groups. Staining was, however, definitely associated with the presence in the dye molecule of 5 or more aromatic rings. This suggested that van der Waals forces of attraction may be responsible for elastic fibre staining both by Direct dyes and the standard elastic fibre stains. Staining of elastic fibres as a side-effect in many procedures is similarly explicable.     

The ultrasensitive silver "protein" stain also detects nanograms of nucleic acids

The ultrasensitive silver staining procedure developed for proteins also stains nanogram quantities of RNA and DNA in polyacrylamide gels. A gradient polyacrylamide gel system is described which separates proteins from 10⁴ to 10⁶ M_r, RNA from 5S to 23S and DNA from 0.4 to 21 Kb. The sensitivity of nucleic acid silver staining in this gel system considerably exceeds that of commonly used DNA and RNA dye-binding stains.     

An Assessment of Routine Chromosomal Staining Procedures in Radioautography

Unlabeled human chromosome preparations were treated with commonly employed chromosome stains as follows: (I) they were stained, destained, coated with liquid emulsion, developed, fixed, and restained; (II) stained and coated directly; or (III) coated and then stained. Of the stains tested, the methylene blue-eosin type (Giemsa, MacNeal's, Wright's) was useful for application after coating, although a similar stain (eosin-Stevenel's blue) caused formation of a heavy precipitate in the emulsion when so used. None of these stains could be employed before coating, however, even though they were removed with acid alcohol prior to dipping, because they caused chemographic grain formation in the emulsion. Aceto-orcein and Feulgen could not be employed after coating because the procedures removed the emulsion from the slides. Safranin was also found to be ineffective for staining coated preparations due to chemical changes caused by the photographic processing. The only stain which did not cause chemography, and hence can be used before coating slides, is aceto-orcein. Since this stain fades during radioautographic processing and cannot be employed after coating, we recommend secondary use of one of the methylene blue-eosin type stains for revisualization of the chromosome spread.     

Simultaneous visualization of immunodetected antigens and tissue components revealed by non-enzymatic histochemical stains.

We explored the possibility of simultaneous application of histochemical and immunohistochemical staining techniques on the same paraffin-embedded human tissue section. Conventional histological stains (PAS, Alcian, Alcian-PAS, Van Gieson, Gomori silver impregnation, and Giemsa) were used in association with a battery of markers (keratins, leucocyte common antigen, S-100 protein, Factor VIII-related antigen) that are widely employed in diagnostic and experimental studies. We found that the best procedure was to perform immunostaining before the histochemical reaction, as this enables all the other possible combinations to be carried out. In addition, several detection systems, such as peroxidase-anti-peroxidase (PAP), alkaline phosphatase-anti-alkaline phosphatase (APAAP), and avidin-biotin complex (ABC), were tested and all gave consistent results. Some minor modifications of the histological staining methods were necessary, but the current immunohistochemical techniques could be used as established. Preliminary findings indicate that immunohistochemistry can be combined with histochemistry techniques by means of a relatively simple procedure whose only disadvantage is the time required to carry out the double staining.