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.     

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.     

Staining Etched Epoxy Resin Sections for Light Microscopy

Staining of etched sections for light microscopy is described. Azan staining was successful after treatment with potassium dichromate and the use of concentrated dye solutions. To remove osmium for hematoxylin-eosin staining, removal by reduction with ferrocene was used instead of oxidation. Highly selective differentiation after hematoxylin staining was achieved using p-toluenesulfonic acid-DMSO. To enhance eosin staining, a 2-bromoethylamine link between eosin and the tissue was used. Ferrocene also facilitated counterstaining of nuclei with hematoxylin after the PAS reaction. Periodic acid-methenamine silver staining was carried out without modification.     

Thin Sections from Unfixed Tissues for Histochemical Staining

Sections were cut from fresh unfixed tissues by means of a microtome provided with an apparatus for the simultaneous cooling of the knife and freezing stage. These sections were of uniform thickness and were found to be very suitable for histochemical staining. Such sections were immersed while still frozen in the fluid which contained the necessary chemicals for a specific technic. After remaining in the fluid for an appropriate time, the sections were put on slides and dried in warm air. The remaining steps were carried out on the slides. Several histochemical procedures (phosphatase, esterase, glycogen) were found to give good results when this technic was used.     

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.     

Elimination OF Distortion and Poor Staining in Paraffin Sections OF Plant Tissues

Three fixing solutions causing least distortion and bright staining of plant tissues are named. Glycerin dehydration causes less distortion than a series of alcohol concentrations; 95% alcohol removes some of the glycerin, sets the protoplasm and improves the staining. Absolute alcohol causes distortion and should be avoided. Pure chloroform, as a paraffin solvent, is followed by brighter staining but more distortion than are the butyl alcohols. A schedule resulting in minimum distortion is given. The results are shown in photomicrographs. Brightest staining follows the use of C. P. iron alum and hematoxylin. The use of a paper cup for very gradual change from one liquid to another and as a labor saver is described.     

An Evaluation of Various Fixing and Staining Procedures for Mitochondria in Plant Root Tissues

Mitochondria were stained intensely by a Regaud iron-hematoxylin procedure for roots fixed in formalin-sublimate or in Helly's fluid. Formalin-sublimate fixation required iodization during the staining sequence, but roots fixed in Helly's fluid were best iodized to remove mercurial precipitates before embedding in paraffin. Both methods required treatment with 1% KOH before immersion in the staining solution to remove RNA and produce pale cytoplasm. A third successful method was to postosmicate methacrylate-embedded roots after fixation in Hermann's fluid. Blackened mitochondria were produced by the postosmication and further staining was unnecessary. Fixation in Regaud's fluid did not give successful stains in any of the three methods tested. A prefixation treatment in quinone did not aid in obtaining sharply stained mitochondria of roots fixed in Bouin's fluid and stained with Heiden-hain's iron-hematoxylin.     

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.     

Elimination Or Reduction of Wrinkles in Semithin Epoxy Sections by Vacuum Drying

Vacuum drying, under appropriate conditions, diminishes the warping and buckling of epoxy semithin sections and enhances visualization with light microscopy. Treatment of sections with chloroform or variations in the drying times or temperatures did not reduce wrinkling.     

Hematoxylin Staining of Tissues Embedded in Epoxy Resins

Sections of 0.5-2 μ thickness are affixed to slides with albumen adhesive, thoroughly dried, and placed in xylene or toluene for 1 hr, then brought through ethanol to water. Sections of tissue fixed in O_sO₄ are treated first in 0.1% KMnO₄, then with 1.0% oxalic acid, and after rinsing, incubated at 60 C for 12-24 hr in hematoxylin (Harris's or Ehrlich's) and counterstained 10-15 min with 0.5% phloxine B. Permanent preparations are made by clearing and mounting in a synthetic resin. The method requires only easily available reagents and is suitable for routine processing of epoxy sections.     

An Adaptable Staining Schedule for Plant Tissues

A general schedule for staining meristematic, maturing, and mature plant tissues is described. Treatment with a dilute aqueous solution of Delafield's hematoxylin is followed with staining in 0.1% safranin in 60% alcohol. Destaining of safranin may be partly accomplished in alcohol and completed by counterstaining with dilute fast green FCF in a xylene and alcohol mixture. Various modifications and adaptations are briefly discussed.     

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.     

Improved Staining Boxes for Fast, Uniform Staining of Ultrathin Sections on Grids

A standard LKB (LKB-Produkter Ab. S161, 45 Bromma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. the staining boxes can be cut out to any required size or shape. the polymethacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant thin polymethacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. for staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. the separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.     

Feulgen Staining of Intact Plant Tissues for Confocal Microscopy

A method was developed to prepare plant structures for confocal laser scanning microscopy by combining Feulgen staining with pararosaniline and embedding in LR White_TM. This procedure preserves intact, delicate structures for three-dimensional imaging without loss from sectioning or squashing, and the slides can be viewed several times without serious photobleaching.     

Feulgen Staining of Intact Plant Tissues for Confocal Microscopy

A method was developed to prepare plant structures for confocal laser scanning microscopy by combining Feulgen staining with pararosaniline and embedding in LR White_TM. This procedure preserves intact, delicate structures for three-dimensional imaging without loss from sectioning or squashing, and the slides can be viewed several times without serious photobleaching.     

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.