Alcoholic Bouin fixation of insect nervous systems for Bodian silver staining. II. Modified solutions

A previously devised synthetic equivalent of 'aged alcoholic Bouin (Duboscq-Brasil) fixative was modified in various ways to discover which of the chemical changes brought about by aging were important in improving fixation and staining. Effects were tested with ventral nerve cord ganglia of the cockroach Periplaneta americana, locust Schistocerca gregaria, and honey bee Apis mellifera. Formation of reaction products, chiefly ethyl acetate and diethoxymethane, seemed to play only a subsidiary role: neither individually appeared essential as long as a sufficient quantity of one or the other was present. In place of diethoxymethane, ethyl acetate concentration could be increased to 25% with little effect on results. Reduction in concentration of two of the original constituents, formaldehyde and ethanol, appeared to be the principal factor in improving fixation. Varying the concentration of each original constituent individually revealed that formaldehyde mainly increased glial staining, ethanol increased tissue shrinkage and reduced overall staining intensity, acetic acid improved preservation, and picric acid decreased glial staining but produced few other effects within a wide range of concentrations, though its omission seriously impaired overall preservation and staining. Varying the ethanol and acetic acid concentrations simultaneously confirmed that they acted in opposite ways. A decrease in ethanol and an increase in acetic acid both improved results. The optimum mixture, 'improved synthetic alcoholic Bouin' (40% formaldehyde 0-15: ethanol 25: acetic acid 5: ethyl acetate 5: diethoxymethane 15: picric acid 0.5: water to 100), gives better preservation and more intense staining, and formaldehyde content can be varied to give the degree of glial staining and more intense staining, and formaldehyde content can be varied to give the degree of glial staining required. Without formaldehyde glial staining is virtually eliminated, while preservation and staining of the neurons appears unaffected. This modification seems to offer a valuable advance in technique.     

Alcoholic Bouin fixation of insect nervous systems for Bodian silver staining. I. composition of 'aged' fixative

Alcoholic Bouin (Duboscq-Brasil) fixative being 'aged' at 60 C to improve tissue preservation and subsequent staining was sampled at various stages to determine its histological effectiveness and chemical composition. Histological performance was tested using ventral nerve cord ganglia of the cockroach Periplaneta americana and the locust Schistocerca gregaria. Chemical analysis was by ultraviolet spectroscopy, thin layer and gas-liquid chromatography, and mass spectrometry. Histological performance improved rapidly during the first 7-10 days and composition changed correspondingly. The rate of change then slowed as a more stable condition was approached. Fully aged solutions, after about 40 days, giving optimum fixation and staining, contained little more than half the amounts of the volatile components (formaldehyde, ethanol, and acetic acid) in the original mixture, together with ethyl acetate and a formal, diethoxymethane, as the principal reaction products, but picric acid content showed little change. Older ('overaged') solutions, fully aged and then kept at room temperature for 1-2 yr, gave poorer fixation and staining and contained still less of the original volatile constituents and correspondingly more of the reaction products. A 'simplified synthetic aged alcoholic Bouin' (15 ml 40% formaldehyde, 35 ml ethanol, 3.5 ml acetic acid, 5 ml ethyl acetate, 15 ml diethoxymethane, 0.46 g picric acid, and water to 100 ml) closely stimulated the performance of the fully aged orthodox fixative without the need for aging.     

A silver impregnation method for embedded central nervous tissue of the rat

A simple and yet reliable silver impregnation method, using potassium ferrocyanide, for demonstrating nervous tissue of the rat central nervous system embedded in paraffin or paraplast is described. The method reported here is compared and discussed with earlier techniques using potassium dicyanoargentate, potassium ferrocyanide and potassium ferricyanide.     

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 Susa. 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 2-5 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.     

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 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 new fixative solution to precede the reduced silver impregnation of arthropod central nervous systems.

Arthropod central nervous tissue is fixed for 1 hr at 20 C in 8% pure formic acid in 1:1 n-butanol/n-propanol prepared immediately before use (FBP), then washed for 15-30 min in 90% ethanol, and embedded in paraffin wax. Impregnation is by modified Ungewitter techniques in which the silver bath is preceded by mercury/cobalt mordanting, or by modified Holmes' methods following similar mordanting procedures. The methods yield high resolution of axons with minimal background staining, while the staining of neuronal somata is suppressed. They succeed with brains of crustacea and Odonata and other difficult materials. Tissues fixed in FBP are hard and require care in sectioning.     

A silver staining method for single-cell gel assay.

The single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains. (J Histochem Cytochem 49:1183-1186, 2001)     

Bodian's silver impregnation of endocrine cells. A tentative explanation to the staining mechanism

We have recently shown that a variety of proteins, including albumin and immunoglobulins conjugated to colloidal gold, strongly binds to certain basic peptide sequences, and neurohormonal peptides. Silver proteinate, used in the classical Bodian's neurohistological procedure, is now shown to bind to the same peptide sequences in cytochemical model systems. In tissue, gastrin cells and glucagon cells have been reported to show strong unspecific immunocytochemical staining and these cell types also stain in the Bodian's procedure. These results suggest that certain types of unspecific immunocytochemical staining and the Bodian's silver staining method may depend upon a common mechanism, involving binding of labelled or aggregated protein to basic and hydrophobic sequences in tissue.     

A method for combined C-banding and silver staining

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.     

A combined silver and acetylcholinesterase method for staining intramuscular innervation.

A combined acetylcholinesterase and silver stain for demonstrating the intramuscular innervation of fresh frozen tissue is described. Intramuscular nerves, subterminal axons, and motor end plates are simultaneously stained brown or black with minimal staining of connective tissue and muscle fibers in longitudinal sections 30-100 mu thick. The method has been applied to fetal and adult rat, porcine, and bovine skeletal muscle. Antemortem and postmortem tissue samples stained equally well. The method facilitates simultaneous appreciation of morphological alterations in nervous and muscular tissues; in clinical and research laboratories alike it is of value when muscle abnormalities which may be related to disorders of nervous origin are studied. Compared with other published procedures this method has shorter time requirements, uses fresh frozen tissue, and displays superior staining characteristics.