Selective Staining of Neurosecretory Material in Semithin Epoxy Sections by Gomori's Aldehyde Fuchsin

The epoxy resin was removed from semithin (1 μm) sections by immersing them for 30 sec in sodium methoxide (Mayor et al., J. Biophys. Biochem. Cytol., 9: 909-10, 1961) and then processed as follows: (1) left for 1-3 hr at 60 C in a mixture of formalin, 25 ml; glacial acetic acid, 5 ml; CrO₃, 3 gm; and distilled water, 75 ml: (2) oxidized 10 min in a 1:1:6 v/v mixture of 2.5% KMnO₄, 5% H₂SO₄ and distilled water: (3) bleached in 1% oxalic acid, and (4) stained for 15 min in aldehyde fuchsin, 0.125% in 70% alcohol, or in a 1% aqueous solution of toluidine blue. The neurosecretory material is selectively stained.     

Improved Staining Procedures for Semithin Epoxy Sections of Plant Tissues

Improved and reliable methods are described for staining semithin sections of plant materials fixed in glutaraldehyde-osmium and embedded in epoxy resins. One-micron sections are fixed to slides, stained with a two-solution hematoxylin procedure or with a methylene blue-azure A combination, counterstained in aqueous safranin O, cleared, and mounted permanently. Basophilic tissue components arc stained gray to black by the hematoxylin and blue or purple by the methylene blue-azure A combination; all wall structures are colored by the safranin. With the procedures recommended, stains am sharp and intense, sections arc flat, wrinkling and loss are held to a minimum, and unsightly precipitates do not form.     

Polychrome Staining of Epoxy Semithin Sections Using Cacodylic Buffer as a Stain Solvent

Many polychromatic stains are in use for epoxy-embedded tissues (Horobin 1983). We should like to report a quick and easy polychromatic staining procedure that we find useful for routine use. Formerly the stain we used was prepared in 20 ml water and 5 ml 96% alcohol, and gave polychromatic staining only at pH 7.4 obtained by the addition of 1 N NaOH. However, the stain gave satisfactory results only for two or three days. We found that stabilization of the staining solution through the use of an ethyl alcohol-cacodylic buffer solvent increased the life of the staining solution. This was convenient because the cacodylic buffer is used in our laboratory as a component of fixatives, and is not prepared specially for the staining.     

A Technique for Fluorescence Microscopy in Semithin Sections

We describe here a procedure to improve contrast and resolution in fluorescence microscopy of sectioned tissues. Tissue fragments were fixed in ethanol-glacial acetic acid, embedded in diethylene glycol distearate, and semithin sectioned. This method maintains tissue antigenicity while preserving the structure of cells and tissues. The thinness of the sections eliminates scattered and emitted light from tissue structures outside the plane of focus. The procedure is simple and quick, and works excellently with fluorescein-conjugated lectins and antibodies.     

Diaminobenzidine as a Myelin Stain in Semithin Plastic Sections

A diaminobenzidine (DAB) stain for myelin in glutaraldehyde fixed, osmicated, semithin epoxy sections is described. One or 1.5 μm sections, dried onto slides, are first etched with a 1:2 dilution of saturated sodium ethox-ide:absolute ethanol, then incubated in 0.05% aqueous DAB with 0.01% hydrogen peroxide. DAB specifically stains osmium fixed myelinated nerve fibers. This permits high resolution light microscopic study of myelinated nerve fibers in semithin sections of tissues that also can be studied by electron microscopy.     

Vacuum Freeze Drying of Frozen Sections for Dry-Mounting, High-Resolution Autoradiography

Specimens no larger than 1.5 × 1.5 × 2 mm were frozen in liquid nitrogen and sectioned, while still frozen, with a refrigerated microtome. The frozen sections were dried in a vacuum, then pressed onto either Kodak NTB10 plates or onto slides which had been coated with Kodak NTB3 emulsion and dried. Radioactive mouse liver was used to test tissue preservation. Intestinal mucosa with Ha-labeled nuclei was used to test the quality of autoradiography. Good cytological detail was preserved in both tissues, with the autoradiographs interpretable at the cellular level.     

Rapid Single-Solution Polychrome Staining of Semithin Epoxy Sections using Polyethylene Glycol 200 (PEG 200) as a Stain Solvent

Rapid, onestep polychromatic staining of 0.75-1.5 μm epoxy sections of glutaraldehyde-osmium fixed tissues can be obtained with mixtures of basic fucbsin and toluidme blue O in alkaline polyethylene glycol ZOO (PEG ZOO). Sections are attached to slides by heating at 100 C for 45 seconds and stained at that temperature for 2-3 minutes with a solution consisting of PEG 200 (50 ml), 0.2 N KOH (0.75 ml), basic fuchsin (1.7 gm), and toluidine blue O (0.3 gm). Red-blue balance and selective staining of different structures can be controlled by varying the amount of toluidine blue added. After rinsing with 10% acetone and rapid drying, sections are covered with immersion oil or mounting medium and a cover-slip. Total time from cutting of a section to finished preparation is less than 6 minutes. This staining solution is stable, does not produce precipitates on the sections, and does not wrinkle or lift the sections from the slides.     

A Method for Making Ribbons of Semithin Plastic Sections

Epoxy resin sections form strong, heat resistant ribbons if, prior to sectioning, contact cement has been painted onto the leading and trailing faces of the block. The forming ribbon floats onto a drop of water held in place by a wax line drawn across the back of the glass knife parallel to the cutting edge. A long trough made from stainless steel tubing is inserted horizontally into the drop, and as the ribbon lengthens it is directed into the trough. The ribbon can be carried in the trough to a hot plate for expansion and then poured onto a slide for mounting. The serial ribbons obtainable by this simple procedure greatly facilitate three dimensional reconstruction of fine tissue structures.'     

Prevention of Negative Chemography in Phenylenediamine Stained Autoradiographs of Plastic Semithin Sections

Negative chemography is the loss of latent image daring autoradiographic exposure, due to reactive groups in the specimen. Tissue fixed with ghuaraldehyde and osmium tetroxide, block stained with ft-phenylenediamme and embedded in Epou for light microscope sections causes intense negative cbemography when autoradiographed by dipping in Ilford K2 emulsion: this cannot be completely prevented by separating section from emulsion by means of a layer of evaporated carbon. Chemical treatment of the sections before autoradiography may reduce the cbemography. Treatment with 1 % hydrogen peroxide for 15 nun reduced it to such an extent that subsequent coating with 5 nm carbon abolished it. Material block stained in this way gave excellent contrast, both for light and electron microscopy.     

Silver Staining of the Nucleolar Organizer Regions (NORS) in Semithin Lowicryl Sections

The one-step silver technique was applied to semithin Lowicryl sections of root meristem cells of Allium cepa and a human tumor cell line (TG cells). In vegetal cells, after 5 min of staining reaction, the Ag-NOR proteins formed ring-shaped structures peripherally within the nucleolus. In animal cells silver granules were distributed over the entire nucleolus. The specificity of the staining reaction was increased by incubation of the sections in NH₄Cl and Schiff's reagent prior to Ag-NOR silver staining.     

Preparing Epoxy Sections of Insect Tissues for Light Microscopy

Sections of aldehyde-fixed and osmium-stained insect tissues embedded in various epoxy resins were affixed to glass slides by use of a slide cover and hotplate combination. A high concentration of solvent vapor over the sections was thus maintained while they dried down on the slides, resulting in excellent flatness and adhesion. Sections were then stained at an elevated temperature with a mixture of equal parts of 3 dye solutions: 1% toluidine blue O, 1% safranin O, and saturated auramine O, all made up in 1% solution of borax in water. The method resulted in excellent differentiation of all insect tissue components including lightly chitinized structures.     

A Solution for Removal of Resin from Epoxy Sections

Development of a resin-dissolving solution for use at low alkali concentrations is described. Crown ether dissolved in dimethyl-sulfoxide produces a superbasic alkoxide anion. A five minute treatment resulted in complete resin removal from kidney biopsy specimens embedded in Epon 812. Specimens were well stained by Loeffler's methylene blue. Periodic acid-methenamine silver and Giemsa stains yielded good results. Application of PAS reaction and subsequent hematoxylin counterstaining was practicable for diagnosis.     

Polychromatic Stains for Thin Sections of Beta Embedded in Epoxy Resin

Thin sections of leaves and anthers of Beta vulgaris L., fixed in glutaraldehyde-OsO₄ and embedded in epoxy resin, were stained with different stains at pH ranges from 5 to 9 at 50 C to select those that provided polychromatic staining of suitable intensity. The thionin derivatives, Azure B, Toluidine Blue O, and polychrome Methylene Blue provided adequate staining, as did the commercially prepared stain Paragon PS 1301. Azure B stain was superior for sugar beet 0.5μ monitor sections: cytoplasm appeared grey; nuclei, blue-gray; nucleoli, blue; chloroplasts, blue-green; primary walls, blue; and secondary walls, light blue. Choice of one of the stains mentioned probably would depend upon the plant material under study.     

Open-Face, Epoxy Embedding of Single Cells for Ultrathin Sections

Intact stamens of Tradescantia were fixed, dehydrated, and infiltrated with an epoxy resin. Each stamen was then put into a drop of resin on a microscope slide, which was transferred to the stage of a dissecting microscope so that individual hairs could be detached from the filament with fine tungsten needles. The detached hairs were transferred to drops of resin ca. 2 mm in diameter (6 or 7 in each of two rows) lying on a slide heavily coated with evaporated carbon. Polymerization was carried out in an oven until the resin attained a degree of viscosity that permitted orientation of the isolated hairs (by using a compound microscope) without their subsequent dislocation. When the small drops of resin had hardened after further polymerization, the positions of the hairs were marked by circumscribing the cells with India ink. The block was pried from the slide after rapid cooling with solid CO₂, and was then trimmed and sectioned. Cells suspended in culture medium were embedded in much the same way; they were centrifuged to obtain a pellet, which was fixed, dehydrated, and infiltrated. A small fragment of the pellet with a little resin was placed on a microscope slide, where the cells were dissociated under a dissecting microscope at ca. 100 × magnification. Individual cells were then picked up with tungsten needles and transferred to droplets of resin on a carbon-coated slide. The subsequent steps were similar to those described for the staminate hairs. Pieces of tissue in the 50-500 μ range were also handled by the foregoing technique. However, after infiltration they were put into large drops of resin on a slide coated with silicone mold-release rather than on a surface coated with carbon.     

Staining of Different Tissues in Thick Epoxy Resin-Impregnated Sections of Human Fetuses

Sections of undecalcified human fetuses, fixed in formaldehyde, embedded in the epoxy resin Biodur E 12 and cut on a diamond-wire saw were stained according to a slight modification of the method described by Laczko and Levai. The sections were immersed in a methylene blue/azure II solution at 90 C for at least 3 min and counterstained with a basic fuchsin solution at the same temperature. Differential staining was as follows: bone stained pinkish; cartilage, violet; collagen fibers, blue-violet; elastic fibers, red and muscle fibers, green-blue. Most other tissues were stained blue-violet against the transparent background of the embedding epoxy resin. Thanks to the distinct and differential staining of each tissue, contrast is sufficient for black and white as well as for color photography.     

A Method to Prevent Wrinkles in Autoradiographic Emulsion when Staining Plastic Embedded Sections with Hematoxylin and Eosin-Phloxine

A procedure was developed which prevents wrinkles in autoradiographic emulsion when sections, embedded in glycol methacrylate, are stained with hematoxylin and eosin-phloxine. Craniofacial tissues labeled with tritiated thymidine were collected and mounted on slides. Slides were dipped in emulsion, stored for one month and developed. The slides were immersed in liquefied celloidin and subsequently stained with a modified hematoxylin and eosin-phloxine procedure. Results showed that the emulsion did not wrinkle and the procedure did not effect the occurrence of labeled cells.