Stain technology最新文献

A method is described whereby ganglioside GM1 can be quantitated directly on thin-layer chromatograms using cholera toxin subunit B conjugated to horseradish peroxidase and visualized with chloronaphthol. Overlay and color development were performed after separating gangliosides on nano-TLC plates, and fixing with polyisobutylmethacrylate. Absolute quantitation was realized using a Shimadzu CS-9000 integrating spectrodensitometer, scanning at 580 nm. A correlation coefficient of 0.98 was obtained in a linear range of detection from 10(-11) to 10(-16) moles. Statistical analysis revealed good reproducibility and over 99% of the added gangliosides remained with the chromatogram during all overlay and washing procedures. By comparison, standard chemical visualization by resorcinol-HCl was linear in the nanomole range with a detection limit of only 10(-10) moles. Since the carbohydrate portion of gangliosides immobilized in this manner is susceptible to the action of enzymes including neuraminidase, this technique can be applied to all structures of the gangliotetraose series.

Since ovarian follicles appear to be randomly oriented with respect to the plane of the section, the method of sectioning and examining follicles at their maximum diameter described here allows direct comparison between oocyte populations of women and small differences can be detected. Re-sectioning for EM allows selected follicles of interest to be examined at a higher resolution.

Cerium-based methods have been used for the demonstration of several phosphatases at alkaline, acid and neutral pH in low temperature acetone-fixed, plastic-embedded sections. At alkaline pH calcium is used as capturing agent and the precipitated calcium phosphate converted to cerium phosphate. At neutral and acid pH cerium is used directly as capturing agent. Cerium phosphate is subsequently visualized using the H2O2-DAB method. A comparison has been made with conventional calcium-cobalt and lead methods. It appeared that calcium-cobalt methods are more susceptible to improvement than lead methods.

A continuing problem in immunogold labeling of 1 microns LR White sections for light microscopy is the lack of adherence of the sections to the glass microscope slides during silver enhancement. A technique is described to overcome this problem using a 2% Formvar solution to coat the glass.

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and other 4,4'-stilbene-2,2'-disulfonate derivatives used as reagents in histochemistry and physiology have been prepared in their E isomeric form, and rearranged to the Z isomers by irradiation with visible light. Infrared, and 1H and 13C nuclear magnetic resonance spectra were recorded for these compounds, and used to establish the chemical structures. In particular, it was shown that the E-isomer of SITS decomposed in aqueous solution by hydrolysis of both the acetamido and isocyano groups yielding a diamine; disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) also decomposed in solution, while disodium 4,4'-dinitrostilbene-2,2'-sulfonate (DNDS) rearranged from the E-isomer to the Z-isomer when solutions were kept unprotected from light. These results indicate that benchworkers should not be surprised when commercial samples of such stilbenes contain large amounts of various types of impurities.

A simple procedure to stain phenols in plant tissues is described. Postfixation with an aqueous solution prepared by mixing 2 cc of 2% osmium tetroxide and 8 cc of 3% potassium iodide yields brilliant visualization of phenol-containing vacuoles in different tissues of plants (e.g., coffee, oak, tobacco and spruce) bearing high concentration of phenolic compounds. Areas bearing phenols become dark gray to black. Chemical experiments demonstrate that osmium-potassium iodide (Os-KI) mixture reacts rapidly with several naturally occurring plant phenols, developing black solutions from which black solids precipitate. Phenols containing omicron-dihydroxy groups react with Os-KI solution more rapidly than other structurally different phenols. Therefore, omicron-dihydroxy units in an aromatic ring seem to function as primary sites of reactivity with the osmium-iodide complexes.

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.

The cellophane technique of La Cour and Faberge has been improved by the use of a booklet of filter paper. The booklet consists of seven squares of filter paper stapled together; the cellophane on which the pollen is germinated is placed between the two top leaves of the booklet and the whole soaked in a sucrose-based nutrient medium for 15 min. This arrangement keeps the cellophane flat as it absorbs medium. The top leaf of the booklet is then removed, the pollen dusted on it and the completed preparation closed in a plastic-wrapped Petri dish. The lower leaves of the booklet keep the cellophane moist for up to 24 hr. Proportions of pollen grains germinating are at least as high as in the hanging drop method; pollen of species that germinate poorly or not all in hanging drops do well in this technique. Because the pollen tubes adhere tightly to the cellophane, staining, observation, and studies of various sorts are facilitated.

The application of Miller's dilute elastic stain followed sequentially by Gill's III hematoxylin and a fast green counterstain produced a reliable and consistent method for differentially staining elastic fibers, nuclei, muscle and collagen in glycol methacrylate tissue sections. Evaluation of different methods of fixation and conditions of staining on animal tissue sections showed that elastic fibers in both perfusion and immersion fixed tissues can be intensely stained. The stability of Miller's elastic stain offers the potential of a commercially available histological stain reagent for coarse and fine elastic fibers in glycol methacrylate tissue sections.