A Hematoxylin and Eosin-Like Stain for Glycol Methacrylate Embedded Tissue Sections

A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5 percent ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075 percent ponceau de xylidine and 0.025 percent acid fuchsin in 10 percent acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.     

Glycol Methacrylate Embedding in Histotechnology: The Hematoxylin-Eosin Stain as A Method for Assessing the Stability of Glycol Methacrylate Sections

Glycol methacrylate (GMA) samples containing inhibitor in the range of 200-300 ppm were included in a standard embedding mixture. The pH of the GMA samples was measured as a 10% solution of the sample in distilled water. The acidity of GMA due to methacrylic acid causes background staining of sections after basic dyes. The concentration of GMA and the amount of impurities such as methacrylic acid (MA) and ethylene glycol dimethacrylate (EDMA) were measured by gas chromatography. Distinct variations in purity were found among five samples of GMA. Sections derived from GMA samples containing more than 2% EDMA showed few, if any, minifolds after staining with hematoxylin and eosin and were more stable in alcoholic and basic solutions; sections from purer GMA showed minifolds and were less stable. Addition of crosslinkers, EDMA or triethylene glycol dimethacrylate (TEDMA) prevented these artifacts. Crosslinkers clearly influence dimensional changes in sections. Addition of crosslinkers to GMA samples containing minimal amounts of MA improved the results. The possibility of obtaining a high quality GMA embedding medium is discussed.     

Glycol Methacrylate Sections of the Crystalline Lens

Sections of the crystalline lens are difficult to prepare because of the hardness of the fixed lens. After paraffin procedures the lens shatters and cracks when cut because the reagents and high temperatures used for infiltration further harden it. Plastic has been successfully used as an embedding medium for other difficult tissues. It allows prolonged infiltration times at room temperature, and provides a firm matrix for tissues containing areas of varying density. However, standard procedures for embedding tissue in plastic do not allow for complete infiltration of the crystalline lens. The purpose of this report is to describe a modification of the glycol methacrylate embedding technique which ensures complete infiltration of the lens. The following protocol was found to produce consistently good 1-5 μm sections of lenses from 10-2O-day-old rats.     

Glycol Methacrylate Sections of Frozen-Dried Tissues for Histofluorescence

Paraformaldehyde-induced fluorescence in frozen-dried tissues survives embedding in glycol methacrylate. After freeze-drying and treatment with paraformaldehyde vapor, tissues to be examined by this technique are immersed in glycol methacrylate and placed in a dessicator which is then evacuated. They are usually left overnight in the dark; next day, the polymerizer is added and the tissues are again left overnight in the dark in the evacuated dessicator; for smaller blocks or certain tissues, these times can be shortened. The blocks are cut on a JB-4 microtome. Sections of 1-10μ can be made readily with a dry glass knife according to standard procedures.     

Application of the Papanicolaou Stain to Paraffin Sections

Routine paraffin sections from tissues fixed either in aqueous formalin, 80% alcohol (with or without 1% trichloracetic acid added), Carnoy's alcohol-chloroform-acetic (6:3:1) and Bouin's fixative were stained as follows: Harris' hematoxylin, 6 min; running water, 2-3 min; ascending grades of alcohol to 95%; orange G, 0.5% and phosphotungstic acid, 0.015% in 95% alcohol, 5 min; 95% alcohol, 2 changes; Papanicolaou's EA36, 2.5 min; dehydration, clearing, and covering in Permount. The results show morphology better than hematoxylin and eosin and the technic is recommended particularly for keratin, which always stains bright orange.     

Improved Techniques Using Giemsa Stained Glycol Methacrylate Tissue Sections to Quantitate Basophils and Other Leukocytes in Inflammatory Skin Lesions

Improved techniques were developed for processing inflammatory skin lesions in glycol methacrylate (JB-4, Polysciences, Inc.) and for quantitating their leukocyte infiltrates by light microscopy: (1) fixation of entire pelts from rabbits, guinea pigs, rats and mice bearing multiple lesions eliminated artifacts due to biopsy and produced uniformly oriented skin sections; (2) adding dimethylsulfoxide and hydrogen peroxide to the Karnovsky-type fixative increased the rate and effectiveness of fixation; (3) the presence of glycerol in the infiltrating methacrylate and the polymerized plastic block improved the sectionability of skin and other tissues; (4) coating slides with JB-4 Solution A prevented detachment of specimens; (5) Giemsa staining at a carefully selected pH provided optimal differentiation of leukocytes from the several species examined, including man. These techniques, which allowed an accurate histologic assessment of inflammatory skin lesions, were especially valuable for quantitating basophils.     

A One-Solution Tannic-Aced-Iron Stain for Plant Tissue Sections

After completing the bulletin on “Actinomycetes in various parts of the potato and other plants” (Lutman, 1945) the author found the beautiful plates in the atlas to Olivier's monograph (1881) on root structure in which the same intercellular inclusions were shown. Olivier stated that he had stained his sections in “tannate of iron”. Attempts were made by the author to prepare and use such a combination but they were unsuccessful owing to the precipitate that was formed.

The formula used by the U. S. government for ink for official use was tried. This combination is composed of tannic and gallic acids with ferrous sulphate and is acidified with hydrochloric acid. When used double strength, as suggested for special blackness and permanence, the stain was very successful on sections of potato roots and tubers. It stained the Actinomyces hyphae very differentially and was decolorized from all other cell organs. Any other stains used dyed also the pectins and the Actinomyces secretions (melanins) but with this iron tannate combination in one solution, the finest hyphae could be seen and photographed. Since hydrochloric acid was used in this stain, such Actinomyces inclusions must be very tannophylic; much more so than any animal intercellular inclusions so far described.     

Synthetic Orcein as an Elastic Tissue Stain

A study was made of various synthetic orceins in an effort to determine the optimal conditions for their use in staining elastic tissue. A simple technic has been developed, based on a modification of the method originally worked out by Taenzer. Orcein is used as a 0.4% solution in 70% alcohol containing 1% HC1. Sections are counterstained with Mallory's borax methylene blue. The solutions are easily prepared and may be used for several months. Although several methods require staining in orcein from 2 to 24 hours, the present method requires staining for only 30 minutes. After several types of fixation some of the dye samples stained collagenous tissue to varying degrees, but this could be diminished by slightly reducing the strength of the staining solution. Differential staining of elastic tissue was particularly specific in tissues fixed in acetone, collagen in these preparations remaining practically unstained by any of the dye samples.     

Alcian Blue Method for Attaching Glycol Methacrylate Bone Marrow Sections to Glass Slides

Detachment of glycol methacrylate sections from glass slides is a common problem during histochemical and immunohistochemical procedures, particularly when large or hard tissue sections are stained and when using caustic solutions, alcohols, or proteases.     

Lymphocyte Membrane Antigens in Glycol Methacrylate Embedded Tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

The Staining Of Acid Fast Bacilli In Sections Of Glycol Methacrylate Embedded Tissues

Acid-fast bacilli can be stained in tissue embedded in glycol methacrylate. Modification of the Ziehl-Neelsen technique, along with changes in the formula of the plastic embedding medium, allow production of 1 to 2 micron sections which retain their integrity throughout the procedure, and within which the bacilli are clearly visible.     

Periodic Acid-Methenamine Silver Stain for Mycobacteria in Tissue Sections

The electron microscope has proven very effective for visualization of various morphological features of bacteria. Cationized ferritin (CF) is a stain commonly used to increase the electron microscopic resolution of bacterial cells, thereby enabling detailed analysis of their morphological and structural features. CF has been useful for microscopic examination of the bacterial capsule, cell wall, S-layer, and various unique morphological structures. In addition, as a cation, CF binds only to negatively charged molecules. Thus, CF has been used to identify sites of anionic charge on the bacterial cell surface, which has led to insights concerning the formation and turnover of bacterial peptidoglycan and the S-layer proteins. As a cation, however, CF may also interact with certain cellular components, causing erroneous interpretation of microscopic results. This review provides a discussion of both the strengths and weaknesses of CF when used as a stain for electron microscopy.     

Enhancement of Histological Detail Using Metanil Yellow as Counterstain in Periodic Acid Schiff's Hematoxylin Staining of Glycol Methacrylate Tissue Sections

Histological detail in sections from tissues embedded in glycol methacrylate was improved by counterstaining PAS/iron-hematoxylin stained sections with a dilute solution of metanil yellow. The addition of the counterstain increases contrast in tissue sections and highlights PAS-positive entities. The staining protocol provides sharp definition of tissue morphology, differentiates cell types and other tissue components and does not produce background staining.     

Application of Glycol Methacrylate Embedding to Herbarium Specimens of Fruits

Aldehyde fixation and glycol methacrylate embedding were applied to herbarium specimens of fruits of the Compositae. Sections 1-2 μm thick were cut with glass knives. Softening was unnecessary and the hydrophilic properties of the resin permitted staining with a number of dyes. Specimens were examined with bright field and polarized light microscopy. The technique gives good structural preservation and resolution even with 81-year-old herbarium material.     

Hematoxylin Toluidine Blue-Phloxinate Staining of Glycol Methacrylate Sections of Retina and Other Tissues

For a detailed study of the developing chick retina a technique has been developed using glycol methacrylate embedding and a hematoxylin toluidine blue-phloxinate stain. After removal of the vitreous body, one half-segment of the eye is dehydrated through graded ethyl alcohols to 95%, infiltrated and embedded in glycol methacrylate, and sectioned at 2 μm. The sections are stained in alum hematoxylin and then in a mixture containing toluidine blue-phloxinate from a stock solution of the dye that has matured for 2-3 weeks. Differentiation is not required and there is only slight staining of the plastic matrix. The quality and clarity of the sections contrasts markedly with that of similarly stained 5 μm paraffin wax sections of the retina. This technique has also been applied to skin, spinal cord, dorsal root ganglia, pancreas and small intestine. The stained sections from these tissues have proved very useful in revealing structural components.     

Combined Lectin Binding and PAS/Alcian Blue Staining in Glycol Methacrylate Sections

Evaluation of cryofixation and paraffin and glycol methacrylate embedding showed that lectin binding was essentially independent of the embedding medium. Fluorescence intensity increased in the following order: glycol methacrylate, paraffin and cryostat sections, The optical resolution increased in the reverse order. Semi-thin glycol methacrylate sections provided satisfactory fluorescence intensities and the best resolution of all embedding techniques applied. Furthermore the lectin treated sections can be stained further using routine histological or specific histochemical methods. The potassium hy-droxide/alcian blue/periodic acid-phenylhydra-zine-Schiff method was used successfully to demonstrate sulfated and nonsulfated sialomucins. Lectins combined with mucin histochemistry allowed visualization of specific sugar residues in the same glycol methacrylate plastic section.