Life Sciences Commons^™

In Situ Hybridization, In Situ Transcription, And In Situ Polymerase Chain Reaction, L. E. De Bault, J. Gu

Scanning Microscopy

In situ hybridization, in situ transcription, and in situ polymerase chain reaction (PCR) are techniques used to detect DNA and RNA sequences within a cell or tissue structure. These three in situ methodologies employ the principles of recombinant DNA to form double-stranded hybrids of DNA-DNA, DNA-RNA, or RNA-RNA. The essence of in situ hybridization (ISH) is the hybridization of a labeled probe to a complementary target sequence, whereas in situ transcription (IST) is the synthesis of complementary DNA incorporating a label directly on the target DNA or RNA within a cell or tissue. In the case of in situ PCR …

Olfactory And Nasal Respiratory Epithelia, And Foliate Taste Buds Visualized With Rapid-Freeze Freeze-Substitution And Lowicryl K11m Embedding. Ultrastructural And Initial Cytochemical Studies., Bert Ph. M. Menco

Scanning Microscopy

Rat olfactory and respiratory epithelia and Rhesus monkey taste buds were studied with rapid-freeze, acetone/0.1% uranyl acetate freeze-substitution and low-temperature Lowicryl K11M embedding, usually in the absence of other chemical fixation and cryoprotection procedures. Ultrastructural features of mucus, cytoplasm, including cytoskeletons, and membranes were better retained than with conventional methods. Some major examples: The mucus of the olfactory epithelium consisted of a single layer; that of the respiratory epithelium had an electron-opaque sol layer surrounding cilia and microvilli below a thin laminated electron-lucent gel layer. Taste-bud pores displayed a foam-like opaque secretory product, resembling the contents of secretory granules within …

X-Ray Microanalysis Of Calcium Containing Organelles In Resin Embedded Tissue, G. Nicaise, I. Gillot, A. K. Julliard, E. Keicher, S. Blaineau, J. Amsellem, J. C. Meyran, M. L. Hernandez-Nicaise, B. Ciapa, C. Gleyzal

Scanning Microscopy

The localization of calcium in cell organelles at the electron microscope level is often achieved through cytochemical techniques, and verified by X-ray microanalysis. Various methods have been used to cytochemically detect calcium or calcium-binding sites : calcium loading, calcium substitution by strontium, barium, or even lead, and calcium precipitation by oxalate, phosphate, fluoride, or pyroantimonate. Their results may have heuristic value, particularly in preliminary studies of poorly known cell types. A complementary and more physiological approach is offered by quantitative measurement of the total calcium content of organelles after cryofixation.

Resin embedding is less demanding than cryomicrotomy and gives better …

Ultrastructural And Functional Effects Of Lipopolysaccharide And Interleukin-2 On Human Nk Cells, Yuan-Hsu Kang, Mitchell Carl, Lorrita P. Watson

Scanning Microscopy

Bacterial endotoxin (lipopolysaccharide, LPS) and interleukin-2 (IL-2) are known to stimulate NK cell mediated cytotoxicity against tumor cells. In the present report we sought to correlate the stimulatory effect of LPS and IL-2 on NK cell activity with ultrastructural changes which occurred as a result of such stimulation. Peripheral blood mononuclear cells (PBMC) were purified from healthy donors by a Ficoll-Hypaque density gradient technique. Leu-11a⁺ NK cells were isolated by flow microfluorometry using a monoclonal FITC conjugated anti-Leu-11a antibody and a FACS II cell sorter. The PBMC were incubated, respectively, with E. coli LPS or recombinant IL-2 (IL-2) for …

Extraneous Background-Correction Program For Matrix Bound Multiple Point X-Ray Microanalysis, W. C. De Bruijn, M. P. C. Van Miert

Scanning Microscopy

A program is described that allows online determination of extraneous background in multiple point X-ray microanalytical matrices. The program is based upon the calculations of the extraneous background for the film (when present), the standard and the unknown by (100 sec.) point analysis. The program searches for a peak-free part of the spectrum in which the calculated value for the extraneous background is about equal to the value in this region of the spectrum (=b_e). Online the contents of this b_e-region is subtracted from an unmanipulated continuum region in the vicinity of the element present in …

Image Analysis And X-Ray Microanalysis In Cytochemistry, W. C. De Bruijn, H. K. Koerten, M. I. Cleton-Soeteman, C. J. G. Blok-Van Hoek

Scanning Microscopy

When cytochemical reaction products are homogeneously distributed within an organelle, point analyses suffice for the quantitative approach. However, quantitative analysis becomes tedious, when the elements in the reaction product are inhomogeneously distributed. Problems arise when elements from two reaction products have to be related to each other, or to endogenous cytological products (ferritin, haemosiderin, calcium, electron dense markers), either topographically or in concentration. When analyzing inhomogeneous/heteromorphical reaction product-containing organelles special attention has to be paid to measure and relate both volume and concentration. In this paper a relative simple structure (eosinophil granules) is chosen to demonstrate that the acquisition of …

Histochemistry Of Colloidal Iron Stained Crystal Associated Material In Urinary Stones And Experimentally Induced Intrarenal Deposits In Rats, Saeed R. Khan, Raymond L. Hackett

Scanning Electron Microscopy

Organic material associated with the calcium oxalate crystals in urinary stones and experimentally induced nephrolithiasis was stained with colloidal iron and analysed by energy dispersive x-ray microanalysis using standard techniques. Iron was positively identified in the stained specimens indicating that some of the organic material is an acidic mucosubstance. The results also indicate that some of the organic material of urinary stones may originate in the kidneys.

Scanning Electron Microscope Cytochemistry Of Blood Cells, D. Soligo, E. De Harven, E. Pozzoli, M. T. Nava, N. Polli, G. Lambertenghi-Deliliers

Scanning Electron Microscopy

The backscattered electron imaging (BEI) mode of scanning electron microscopy (SEM) has been applied to study various histo-cytochemical reactions in biological specimens since the early seventies. Due to numerous, recent technical improvements the BEI mode of SEM now belongs to the routine of many SEM laboratories.

For cytochemistry, BEI has been mainly used to: visualize intracellular structures and organelles; recognize the different cell types in heterogeneous populations or tissues; study the correlations between enzymatic activities and cell surface features.

We have evaluated the most relevant results obtained in the study of blood cells and the possible future applications of these …

Integration Of X-Ray Microanalysis And Morphometry Of Biological Material, W. C. De Bruijn

Scanning Electron Microscopy

It was investigated how to extract both morphometrical and X-ray elemental information from scanning electron microscopical (SEM) or scanning transmission electron microscopical (STEM)-images and how to integrate these two information streams either on line or off-line after storage.

Cytochemical reaction products in cell organelles in ultrathin sections are the biological structures of interest. In such organelles four different situations can be met: morphologically the structures are homomorph or heteromorph; chemically the elements are distributed either homogeneously or heterogeneously. A new program has been proposed and described, which permits determination of both the area and the mean net-intensity value of chemical …

Immuno-Cytochemistry With Backscattered Electrons, D. Soligo, E. De Harven, M. T. Nava, G. Lambertenghi-Deliliers

Scanning Electron Microscopy

Some cytochemical reaction products are visible inside the cytoplasm of cells observed with the scanning electron microscope (SEM) using the backscattered electron imaging (BEI) mode. Methods can be utilized whenever they result in the deposition of heavy metal, like silver, lead or osmium at the sites of the enzymatic reaction.

More recently the BEI mode of the SEM has been demonstrated to improve the detection of immunogold labeled cell surface antigens. Colloidal gold particles, 40 to 15 nm in diameter can be efficiently used for immuno-specific labeling. Moreover, cytochemical reactions can be applied to previously immunogold labeled cells, therefore combining …