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PubMedDate not supplied

Transverse sectioning of plastic-embedded immunolabeled cryosections: morphology and permeability to protein A-colloidal gold complexes.

In order to provide data for meaningful interpretation and quantitation of immunogold labeling on cryosections their morphology and permeability to protein A-gold were evaluated: We studied plastic sections of immunogold-labeled ultrathin and semithick cryosections cut perpendicular to the original cryosection plane. Various soluble and insoluble antigens in different specimens (hemoglobin and histone H5 in chicken erythrocytes, tubulin in Leishmania cells, and outer membrane protein OmpA in Escherichia coli) were fixed with glutaraldehyde-formaldehyde, formaldehyde, or periodate-lysine-paraformaldehyde and incubated with specific antibodies and protein A-gold of different sizes. The cryosection surface may be rough or smooth depending both on the sectioned material and on dehydration and drying artifacts or possibly on the cutting process itself. Well-preserved sections are capable of withstanding considerable deformation without showing clefts or cracks. If the sectioned specimen is sufficiently fixed, protein A-gold is not able to enter the IgG-labeled sections significantly but follows surface irregularities. However, gold particles can be detected within visibly damaged sections.

Animals↗
PubMed1975

A rapid plastic embedding technique for preparation of three-micron thick sections of decalcified hard tissue.

A 24 hour start-to-finish method is described for the preparation of three-micron-thick sections of decalcified hard tissues. Following acetone dehydration, the tissue to be embedded is infiltrated under vacuum with a series of graded clearing solutions which approach the content of the final methyl methacrylate mixture. After overnight in a 35 C oven, the plastic is polymerized by four hours heating at 42 C. Three-micron-thick sections are then easily prepared by using a Jung microtome for high resolution histologic or detailed autoradiographic procedures.

Animals↗
PubMed1990

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step.

We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([3H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [3H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [3H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.

Animals↗
PubMed1982

Immunofluorescent staining of plastic-embedded renal tissue.

Human renal tissues were fixed in cold 4% paraformaldehyde and 0.2% picric acid, embedded in glycol methacrylate (GM), and sectioned at a thickness of 1 to 8 micrograms. Sections were incubated for three hours with nonspecific protease, stained with fluorescein isothiocyanate-conjugated goat antihuman serum samples, and examined by immunofluorescent microscopy. The immunofluorescent staining was compared with that in cryostat sections of the same tissues prepared and stained by conventional methods. The GM-embedded sections showed consistent staining of antigens, comparable with that in the corresponding cryostat sections, and better preservation of tissue structure than in cryostat sections. The antigens in tissues processed by this method are probably stable for long periods.

Antigens↗