Stain technology最新文献

The classic Mallory-Cason staining procedure has been modified for application to sections "on tape" obtained from large deep frozen tissue specimens. These 20 microns cryosections are collected on tape from a large heavy duty cryomicrotome. The stained sections provide anatomical details that are not revealed by other techniques. The merit of this procedure is found in the support of modern medical modalities, both for research and educational purposes.

Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an asymmetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells.

A method for decalcification end point determination of mineralized tissue is described. The calcium content of the decalcification solution was determined colorimetrically with a "continuous automatic analyzer" with a high degree of accuracy. The end point method used has been tested on two decalcification methods, 5% nitric acid with or without ultrasonic treatment. The results suggest it is possible to quantitate the decalcification process.

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 stomata 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 cytoplasmic 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.

Immunohistochemical demonstration of the thymidine analogue bromodeoxyuridine (BrdU) in regenerating cells was useful in determining the size and location of the wounded areas (epithelial and submucosal) during regeneration of the hamster tracheal epithelium, at times late in the healing process (72-144 hr postinjury) when the wound sites and their boundaries were not recognized with certainty in conventionally stained paraffin sections. Cells distant from the wound sites remained unlabelled. The success of this method resulted from prolonged exposure to BrdU released over several hours from a 25mg tablet implanted subcutaneously at 24 hr postwounding at the time when DNA synthesis and cell proliferation are maximal. This simple technique promises to be useful in determining the size and location of wound sites with application to a wide variety of organs and tissues in studies of repair and healing.

A technique for microwave fixation of inflated rat lung is described. Conventional intratracheal fixation with instillation of fixative into the airways at a constant pressure results in pressure artifacts as well as flushing and disruption of cells and exudates. Microwave fixation fixes these elements in situ without disruption and thus is valuable when evaluating the distribution of inflammatory infiltrates. Exudative pneumonitis was produced in the rat using intratracheal instillations of either endotoxin or silica and comparisons were made between histologic sections fixed using either standard formalin fixation or microwave fixation.

After staining with dilute solutions (0.1 mg/ml in distilled water) of commercial carmine, a strong reddish orange fluorescence was observed in nuclei from cell smears and frozen and paraffin tissue sections. Optimal exciting light was 436 nm (violet-blue) or 450-490 nm (blue). Compact chromatin from interphase nuclei, mitotic and meiotic chromosomes and the kinetoplast of Trypanosoma cruzi showed the highest fluorescence, while the basophilic cytoplasm appeared weakly fluorescent. No emission was observed in cartilage matrix, mast cell granules or goblet cell mucin. This selective method could be valuable in microscopic and cytochemical studies on chromatin because the carmine fluorescence is stable and preparations can be dehydrated and mounted permanently without changes in the fluorescence pattern.

A trichrome staining technique using safranin-indigo-picrocarmine (SIPC) can be used to distinguish the various stages of the cell cycle in onion root tip. When the tissue was fixed first in formalin followed by picric acid and stained in SIPC, a clear differentiation of interphase nuclei into four color classes, viz., green, orange, red and brown can be recorded. Replacing crystal violet for safranin produces a similar pattern of differentiation of interphase nuclei into green, light blue, blue and deep blue. Autoradiographic study using 3H-thymidine as a DNA precursor demonstrates the reliability of the SIPC staining technique. All the orange and red nuclei are found to be labelled and therefore are in S phase of the cell cycle. Almost all the green nuclei are unlabelled and may be assigned to G1. The larger brown nuclei which are mostly unlabelled can be considered in G2 phase.

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.