摘要:

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5–700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5–539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.

ISSN:1052-0295    

DOI:10.3109/10520299409106273

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

In quantitative ultrastructural studies using colloidal gold immunocytochemical techniques, labeling intensities vary according to the size of the probe used. Using postembedded indirect two-sided double labeling and single labeling protocols, the labeling characteristics of four antigens were studied using two probe sizes commonly used in double labeling studies. It was determined that the labeling intensity variation resulting from the use of different probe sizes was unpredictable after correcting for the increased probe size alone. It was possible, however, to obtain comparable labeling densities by first determining the labeling intensities for each probe size with its antigen in single label studies on serial sections and using the same procedure as the double labeling studies. A probe size correction factor for each antigen was calculated from these data. This factor was used to obtain comparable measurements of the relative abundance of each label.

ISSN:1052-0295    

DOI:10.3109/10520299409106274

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

A technique utilizing microdissection by ultrasonication was applied to scanning electron microscopy of chick embryos during the first three days of incubation. Using a tank cleaner operating at 80 kHz, whole embryos immersed in pure acetone were sonicated until fragmentation became evident. At 12 hr incubation disintegration occurred by one second or less. At 18 hr, three sonic bursts of one second each produced only partial fragmentation. All three germ layers retained their original relationships to each other. During the second day of incubation, large pieces of integument were removed and somites began to microdissect after 10–20 seconds of sonication. Late in the third day of incubation, sonication for 1 min or more was required to produce significant microdissection. Living embryos exposed to 0.1% collagenase for 10 min prior to standard fixation fragmented in a different manner. Lamellipodia and filopodia were most sensitive and were largely destroyed. The three major germ layers (ectoderm, endoderm, mesoderm), however, retained their structural integrity and original relationships to each other. Factors contributing to the results reported here include: 1) extracellular fibrils of varying chemical composition, 2) primitive cell junctions, 3) biomechanical stability in the nonfibrillar portions of the extracellular matrix, and 4) effects of technical procedures performed prior to sonication. Sonicated tissues of early embryos reveal features that are difficult to demonstrate in other ways and may be unrecognized in conventional preparations.

ISSN:1052-0295    

DOI:10.3109/10520299409106275

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

Whole mounts of mouse oocytes and embryos are useful for observing intracellular structures while preserving morphological integrity. This method is inconvenient for rapid processing of a large number of specimens because washing each specimen in a protein-free solution is required prior to transfer into the fixative. We have developed a new fixative which does not cause protein precipitation which can be added directly to the culture medium. Specimens can be preserved in culture dishes for at least one month, and processed for cytological observation at a convenient time. When stained with hematoxylin, details of cellular structures such as nuclei, nucleoli, chromosomes and spindle microtubules can be observed while maintaining the organization of the organelles.

ISSN:1052-0295    

DOI:10.3109/10520299409106276

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules,p-phenylenediamine (PPD),n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.

ISSN:1052-0295    

DOI:10.3109/10520299409106277

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

Histochemical staining of the granules of eosinophilic granulocytes and subsequent blockade of the reaction by alkaline benzil was strongly suggestive that in its purified form, the diazo dye naphthalene black reacts with tissue sites containing high concentrations of arginine residues. Computer graphics modelling indicated that the sulfonate group of the dye reacts electrostatically with the guanidino functional group of arginine. This acid-base type reaction likely has a stoichiometric ratio of 2:1 in favor of the amino acid.

ISSN:1052-0295    

DOI:10.3109/10520299409106278

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

We have developed a colorimetric method for evaluating the number of osteoblastic cells in culture without destroying the cells. This assay is based on the staining of basophilic cellular compounds with methylene blue. The dye bound by the cells is released at low pH and measured in a spectrophotometer at 662 nm. Linear correlations exist between the absorbance measured by the methylene blue assay and the number of cells seeded, the total cellular protein content, and thymidine labeling. This colorimetric method has the advantage of preserving cell integrity. After destaining, scanning electron microscopy can be performed on well preserved cell morphology.

ISSN:1052-0295    

DOI:10.3109/10520299409106279

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

A simple and rapid method for light microscopic in situ hybridization on cytospin preparations is described and demonstrated for detection of viral nucleic acid in a virus-infected cell line. Cells were fixed by acetone followed by chloroform, denatured by heat, hybridized at 37 C, and hybridized sites detected with a multiple step procedure (primary anti-biotin antibody, biotinylated second antibody, streptavidin-peroxidase). This method can be used for screening studies at the light microscope level, and offers a useful and simple way to determine optimum hybridization conditions for subsequent electron microscopic investigations.

ISSN:1052-0295    

DOI:10.3109/10520299409106280

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

Methylene blue and neutral red were selected for staining mast cell granules by supravital injections. A new technique was applied for embedding in paraffin and Araldite®without dislocation or loss of dye. Stabilization and electron microscopic identification of the dyes were achieved by transforming them into electron-dense precipitates using phosphomolybdic acid dissolved in a paraformaldehyde-glutaraldehyde mixture to preserve the ultra structure of the tissues. It was found that in general the intensity of the light microscopic staining correlated directly with the electron density. Closer study revealed that not all cytoplasmic granules exhibited the same strong affinity for the cationic dyes. Furthermore, differences in dye distribution were observed within the granules themselves. The difference in the staining pattern can be explained by the heterogeneous occurrence of the anionic residues. Because of its high sensitivity and relatively low toxicity, the method described here is well suited for detecting the binding sites of organic cations in tissues under supravital or vital conditions

ISSN:1052-0295    

DOI:10.3109/10520299409106281

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

Specimens of rabbit liver were fixed for various periods up to 6 days in buffered14C-formaldehyde. Binding of the isotope reached a plateau after fixation for approximately 24 hr; the half-maximal binding level was reached after approximately 100 min. Formaldehyde binding at 37 C was faster than at 25 C, and faster at pH 7.0 than at pH 4.0. During rinsing of the fixed tissue in water for up to 26 days there was a progressive decrease in isotope content to 10–20% of the pre-rinse level, indicating that formaldehyde fixation is a reversible process.

ISSN:1052-0295    

DOI:10.3109/10520299409106282

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor