Immunohistochemical Detection of DNA Replication in Mouse Uterine Cells by Bromodeoxyuridine Labeling of Wax- and Resin-Embedded Tissue Sections

TO apply the bromodeoxyuridine (BrdU) labeling method using a monoclonal antibody to the study of cell proliferation in the mouse uterus, methods of fixation and embedding of tissues and of immunofluorescent staining were compared in terms of the rate of detection of labeled cells and specificity and stability of fluorescence obtained. BrdU was administered intravenously 2 hr before death and uterine blocks were embedded in polyester wax and Technovit resin after fixation in formalin and periodate-lysine-paraformaldehyde, respectively. The indirect method with anti-BrdU and fluorescein isothiocyanate (FITC) conjugated antimouse IgG antisera and the direct method with FITC conjugated anti-BrdU antibody were applied to both wax- and resin-embedded sections. Labeled and total cells were counted in luminal and glandular epithelia and stromata adjoining them. Counterstaining with hematoxylin for counting total cells produced intense fluorescence over the whole of resin sections and made counting of labeled cells impossible. On wax sections, on the other hand, the results were satisfactory, although the number of labeled cells detected was decreased slightly. In wax sections fluorescence due to nuclear incorporation of BrdU in the indirect method could be easily distinguished from the cytoplasmic or extracellular emission seen in some cells by its location and characteristic color. In resin sections, however, more careful observation was needed since the second antibody used in the indirect method cross-reacted with IgG in eosinophils and produced cyctoplasmic fluorescence of the same color. By the indirect method greater numbers of labeled cells were detected in wax sections than in resin sections. The difference was distinct in tissues with extensive cell proliferation. By the direct method the fluorescence obtained was weaker and apt to fade more quickly than that obtained by the indirect method; use of the direct method reduced the number of labeled cells detected in both wax- and resin-embedded sections.     

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.     

Rapid Preparation of Thick Sections of Fleshy Plant Tissues

Methods are described for permanent micro-slide preparations of soft, large-celled plant tissues such as ripe fruit. Thick sections (200-800 [t) cut on a sliding microtome are aspirated in an aqueous killing agent; after fixing and washing, the sections are dehydrated and cleared in an alcohol-xylene series. Infiltration with 20, 30, and 40% solutions of mountant prior to mounting the sections is necessary to avoid too abrupt changes in the cleared tissues. Several staining methods have been successfully used for different purposes. The final preparations showed nearly perfect preservation of intact cells and intercellular spaces in their 3-dimension-al structure.     

Controlled Formaldehyde-Catecholamine Condensation in Cryostat Sections to Show Adrenergic Nerves by Fluorescence

Three sets of sections of freshly removed tissue are cut at 18 μ in a cryostat and dried on slides for 1.5 hr over P₂O₅. Each set of sections is incubated with a differently hydrated paraformaldehyde (prepared by storing paraformaldehyde powder over 21%, 25% or 28% aqueous H₂SO₄ for 1 wk) at 80 C for 1 hr before being mounted in glycerol and viewed with a fluorescence microscope. At least one set of specimens shows optimal fluorescence. The entire procedure from removing the tissue to observing fluorescence microscopically is accomplished readily within 4-8 hr. Adrenergic axons in the medial muscle of the cat nictitating membrane, the myometrium of the cat uterus and the adventitia of arterial vessels in rat pancreas are demonstrated.     

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.     

Quantitative Histochemistry: Histologic Sampling of Sections of Frozen-Dried Tissues

Microchemical techniques are available for analyses of many enzymes and other components of tissue for which stains are not available. This paper describes techniques of freeze drying and histological sampling of certain cell groups or structures free from surrounding stroma, fat and other types of cells. Quantitative data can be obtained for many enzymes in 0.5 to 5 μ samples of freeze-dried tissue. For example, glucoses-phosphate dehydrogenase activity was measured and compared in the breast alveoli of mice in estrus, diestrus, gestation and lactation.     

Quantitative Histochemistry: Histologic Sampling of Sections of Frozen-Dried Tissues

Microchemical techniques are available for analyses of many enzymes and other components of tissue for which stains are not available. This paper describes techniques of freeze drying and histological sampling of certain cell groups or structures free from surrounding stroma, fat and other types of cells. Quantitative data can be obtained for many enzymes in 0.5 to 5 μ samples of freeze-dried tissue. For example, glucoses-phosphate dehydrogenase activity was measured and compared in the breast alveoli of mice in estrus, diestrus, gestation and lactation.     

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.     

Staining Residual Lipids in Ultrathin Sections of Tissues Embedded in Polyester Resin

Rat suprarenal glands fixed in Palade's 1% OsO₄, buffered at pH 7.7 with veronal-acetate, to which 0.1% MgCl₂ was added, were embedded in Vestopal-W and sectioned at 0.2-1 µ. The sections were attached to slides by floating on water, without adhesive, and drying at 60-80° C, placed in acetone for 1 min and then treated with the following staining procedure: Place the preparation in a filtered solution of oil red O, 1 gm; 70% alcohol, 50 ml; and acetone, C.P., 50 ml; for 0.5-1 hr. Rinse in absolute ethyl alcohol; drain; counterstain with 0.5% aqueous thionin for 5 min; rinse in distilled water; drain; stain in 0.2% azure B in phosphate buffer at pH 9, for 5 min. Dry and apply a drop of immersion oil directly on the section. The preparations are temporary. Ciaccio-positive lipids, rendered insoluble by OsO, fixation, stained red to ochre.     

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.     

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.     

Differential Staining of Calcified Tissues in Plastic Embedded Microtome Sections by A Modification of Movat's Pentachrome Stain

Movat's pentachrome I stain has been adapted and modified as a stain for Undecalcified bone sections. After embedding in methyl methacrylate, this procedure yields consistently good results, with an excellent and colorful contrast between mineralized and unmineralized compartments of both cartilage and bone. in addition, osteoblats, osteoclasts, and other cells and tissue components can easily be differentiated. the staining properties of the lacunar wall surrounding the osteocytes are considered to reflect various states of osteocytic activity. the method is especially useful for the study of bone growth and bone repair, and as a stain for conventional histomorphometry and computer-assisted image analysis in bone biopsies.     

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.     

Retention in Situ and Spectral Analysis of Fluorescent Vacuole Components in Sections of Plant Tissues

The contents of plant vacuoles vary in different organs and with the health of the plant, but little is known of the cell-to-cell distribution of soluble organic compounds within plant tissues. Soluble fluorescent phenolic compounds can be immobilized in plant tissues using an anhydrous freeze-substitution and resin embedment process. The vacuolar fluorescence can be characterized in fluorescence photomicrographs for variations in color and intensity, or more quantitatively with spectra obtained using a microspectrofluorometer. This is demonstrated here in freeze-substituted roots and leaves of soybean. Excitation and emission spectra of individual vacuoles can be compared with spectra of pure compounds to form profiles of the varied phenolic contents of plant vacuoles. Such analyses will add an important anatomical dimension to the study of plant defense and stress responses.     

Sporopollenin formation in the ascospore wall of Neurospora crassa

Chemical extraction procedures have shown that sporopollenin is a major component of the ascospore wall of Neurospora crassa and Neurospora tetrasperma. The sporopollenin is about 12% total dryweight of ascospores, and is localised in the ribbed perispore layer. Radioactive β-carotene is efficiently incorporated into the sporopollenin, and it is suggested that carotenoids are the natural precursors that are polymerised in the perispore. However, three carotenoid-deficient mutants of N. crassa produced perispores of normal appearance and properties.