Stain technology最新文献

Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.

The fluorescent DNA probes DAPI and Hoechst 33258 produce superior images to the traditional acetocarmine stain of the small chromosomes of the woody shrub Thryptomene calycina at all stages of microsporocyte meiosis and microspore mitosis. Hoechst 33258 was slightly superior to DAPI because of reduced background fluorescence. Binding with the DNA-specific probes required a fixative containing chloroform to remove autofluorescent materials, a pretreatment with acetic acid and a pH of least 6 during treatment. The nucleoli did not fluoresce after treatment with DAPI or Hoechst 33258. Superior resolution of chromosomes after treatment with the fluorochromes enabled easy determination of the haploid number at metaphase I, metaphase II and at metaphase of the microspore mitosis.

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.

Four fluorescent brighteners (Fluorescent Brightener 28, Fluostain 1, Fluostain II and Cellufluor) were examined with respect to their binding affinity, toxicity (their ability to stunt growth), and teratogenic effects on the red alga Antithamnion kylinii. Maximum binding occurred with FB-28 and F-II but these stains showed the greatest inhibition of growth when plants were exposed to concentrations of 0.01% for 30 min. Filaments incubated in low stain concentrations (0.0005%) showed cell abnormalities with all stain types, with FB-28 producing the most extreme deformations of both intercalary and apical cells. The experiments suggest that extensive experimentation is required to develop protocols for vital cell wall stains that minimize toxicity and maximize binding.

A combined method for clearing soft tissues, staining cartilage and bone, and injecting the vascular system of small mammals was developed using Mus musculus (house mouse). Mammalian muscle tissue remains milky or even opaque after "clearing" by previous techniques due to the relatively high content of intramuscular fat. A method employing chloroform-ethanol successfully renders soft tissues of mammalian specimens translucent without damaging or bleeding color from the latex injected in the circulatory system. Resulting specimens yield an excellent view of the skeletal system and the injected vascular system without obstruction by opaque tissues or disruption by physical removal of connective tissue.

An inhibition reactivation technique was used for histochemical staining of human skeletal muscle sections. Myofibrillar ATPase activity was inhibited by sodium hydroxymercuribenzoate (2.5 mM in 0.1 M Tris-HCl buffer, pH 7.2-7.5, 30 min) and successively reactivated by cysteine which was added to incubation solution (10 mM cysteine-HCl, 2.5 mM ATP-disodium salt, 50 mM potassium chloride and 27 mM calcium chloride in barbital buffer, pH 9.4, 35 min at 37 C). This technique allows the distinction of three fiber categories with different staining intensities in single cross-section. Dark, intermediate and light fibers correspond to IIB, I, and IIA types, respectively. Storage of air dried sections in the freezer at -20 C for one month had no influence on staining characteristics.

Myeloma plasma cells were double stained using peroxidase and alkaline phosphatase labelled monoclonal anti-BrdU and anti-intracytoplasmic immunoglobulins. Samples were methanol fixed; DNA was denatured with formamide. The results allowed easy identification of plasma cells, their cytological examination and the calculation of percentage of plasma cells in S phase. Good correlation was found with the labelling index obtained with tritiated thymidine.

The effect was examined of the chemical decomposition of the potassium stain sodium hexanitrocobaltate (III) (SHC), on its ability to produce stain granules of consistent size that could be used to estimate the K+ contents of stomatal guard cells. Stomata in detached epidermis from leaves of Vicia faba (fava bean) were stimulated to accumulate K+ by treating them with fusicoccin. Stomatal apertures and the fraction of guard cell area covered by K+ precipitate granules (K+ score) were measured by digitizing photographic enlargements, and K+ scores were correlated with the age of stain that had been stored either in open or closed containers. The ability of stain aged in open containers to produce consistent fractional cell coverage was compared to 1) the ability of identically treated stain to precipitate K+ from solutions of KCI, and to 2) the kinetics of decomposition of SHC. It was found that the fractional coverage of guard cells of stomata opened to the same apertures decreased with a first order rate constant of 2.3 x 10(-5)/sec. The mass of precipitate formed by treatment of KCl solutions was unchanged for 2 hr after initial preparation of the SHC, and decreased thereafter with a first order rate constant of 1.0 x 10(-5)/sec. When stored in tightly sealed containers, nearly 100 hr were required for an occasionally opened bottle of SHC to decay to the same efficacy as a solution left open to the air for 8 hr.

Red and black spruce and their hybrids can be determined by morphological indices; however, the criteria are somewhat subjective and increasingly difficult to use at higher elevations. Although the chromosome number is identical (2n = 24), red spruce has twice as much nuclear DNA (48 pg) than black spruce (24 pg) and thus the species and their hybrids can also be separated by cytophotometry. This is relevant to spruce decline studies because black spruce is much more resistant to high elevation environmental stresses, both natural and anthropogenic. It also has implications for the effect of climatic changes on the composition of high elevation spruce-fir forests because red spruce can outcompete black spruce under more mesic conditions. Four elevation transects sampling spruce on the east and west sides of Mount Washington (New Hampshire) and Camels Hump (Vermont) and a single transect on the southwest side of Whiteface Mountain (New York) were made to investigate the degree of hybridization and introgression between these two species. A positive correlation was found between increased elevation and increased black spruce genes on Mount Washington and Camels Hump. Pure black spruce was found on Mount Washington from 1356 m to 1582 m. No pure black or red spruce was found on Camels Hump although the proportion of red spruce alleles was significantly greater on Camels Hump. All trees sampled at all elevations on Whiteface Mountain were pure red spruce. Thus the proportion of black spruce alleles in high elevation spruce populations decreases from east to west. This closely parallels the increase in spruce decline which increases from east to west.