Life Sciences Commons^™

Immunocytochemistry By Electron Spectroscopic Imaging Using Well Defined Boronated Monovalent Antibody Fragments, M. M. Kessels, B. Qualmann, W. D. Sierralta

Scanning Microscopy

Contributing to the rapidly developing field of immunoelectron microscopy a new kind of markers has been created. The element boron, incorporated as very stable carborane clusters into different kinds of peptides, served as a marker detectable by electron spectroscopic imaging (ESI) - an electron microscopic technique with high-resolution potential.

Covalently linked immunoreagents conspicuous by the small size of both antigen recognizing part and marker moiety are accessible by using peptide concepts for label construction and their conjugation with Fab' fragments. Due to a specific labeling of the free thiol groups of the Fab' fragments, the antigen binding capacity was not …

Labeling With Nanogold And Undecagold: Techniques And Results, James F. Hainfeld

Scanning Microscopy

A significant new development in gold labeling for microscopy has been achieved through the use of gold cluster compounds that are covalently attached to antibodies or other probe molecules. These unique gold probes are smaller than most colloidal gold conjugates and exhibit improved penetration into tissues, higher labeling densities, and allow many new probes to be made with peptides, nucleic acids, lipids, drugs, and other molecules. A new fluorescent-gold conjugate is useful for examining localization by fluorescence microscopy, then visualizing the same label at the ultrastructural level in the electron microscope.

Pre-Embedding Staining Of Single Muscle Fibers For Light And Electron Microscopy Studies Of Subcellular Organization, Evelyn Ralston, Thorkil Ploug

Scanning Microscopy

Skeletal muscle fibers are large, multinucleated cells which pose a challenge to the morphologist. In the course of studies of the distribution of the glucose transporter GLUT4, in muscle, we have compared different preparative procedures, for both light (LM) and electron microscopy (EM) immunocytochemistry. Here we show that pre-embedding staining of single teased fibers, or of single enzymatically dissociated fibers, has several advantages over the use of sections for observing discrete patterns that extend over long distances in the cells. We report on an optimization study carried out to establish fixation and permeabilization conditions for EM immunogold labeling of the …

Problems In Preparation Of Chromosomes For Scanning Electron Microscopy To Reveal Morphology And To Permit Immunocytochemistry Of Sensitive Antigens, A. T. Sumner

Scanning Microscopy

Although much information about chromosome structure and behaviour has been obtained using light microscopy, greater resolution is needed for a thorough understanding of chromosome organisation. Scanning electron microscopy (SEM) can provide valuable data about these three-dimensional organelles. The introduction of methods using osmium impregnation of methanol-acetic acid-fixed chromosome spreads revolutionised matters, producing life-like images of chromosomes. Nevertheless, it became clear that osmium impregnation introduced various artefacts, although the resulting images were still useful. Methanol-acetic acid-fixed chromosomes are, in fact, flattened on the glass substratum, and the 3-dimensional appearance obtained after osmium impregnation is the result of swelling during this process. …

Pathological And Immunocytochemical Changes In Chronic Calcium Oxalate Nephrolithiasis In The Rat, R. De Water, E. R. Boeve, P. P. M. C. Van Miert, C. P. Vermaire, P. R. W. A. Van Run, L. C. Cao, W. C. De Bruijn, F. H. Schroder

Scanning Microscopy

In the present study, we exposed rats to a crystal-inducing diet (CID) consisting of vitamin D3 and 0.5% ethylene glycol (EG), and we investigated histologically the kidney damage induced by the deposition of calcium oxalate (CaOx) crystals. After 28 days, 50 % of the animals had renal CaOx crystals, of which 60% also had small papillary stones. Most crystals were present in the cortex. The occurrence of these crystals coincided with morphological and cytochemical changes: glomerular damage, tubular dilatation and necrosis, and an enlargement of the interstitium. The number of epithelial and interstitial cells positive for the proliferating cell nuclear …

Low Temperature Techniques As A Tool In Plant Pathology, Sigrun Hippe-Sanwald

Scanning Microscopy

In plant pathology, low temperature preparation techniques now appear to be feasible methods to stabilize the dynamic ultrastructure of the host-(plant)-pathogen (fungi) interaction for an analysis by transmission electron microscopy. A well defined ultrastructure of small organisms (fungi) and large biological samples such as plant material and as well as the plant-pathogen (fungus) infection sites are presented. The mesophyll tissue of Arabidopsis thaliana is characterized by homogeneously structured cytoplasm closely attached to the cell wall. Infection sites of stem rust (Puccinia graminis f. sp. tritici) on primary leaves of wheat (Triticum aestivum) and powdery mildew (Erysiphe …

Comparative Analysis Of Patch Lesions In The Chick Inner Ear Following Acoustic Trauma: Optical Versus Scanning Electron Microscopy, H. J. Adler, J. Mantooth, Y. Raphael

Scanning Microscopy

Neonatal chicks were exposed to an octave band noise with a center frequency of 1.5 kHz at 116 dB SPL for 4 hours. Seven days following overstimulation, the birds were sacrificed. Their basilar papillae were re-moved, fixed in 4% paraformaldehyde, and processed in two steps. First, the ears were immunostained with a supernatant of mouse anti-tectorial membrane antibodies, followed by a diaminobenzidine process. Examinations of the papillae under an optical stereo microscope revealed a patch site with a partially regenerated tectorial membrane (referred to as the honeycomb).

After the optical studies, the same ears were post-fixed in 1% osmium tetroxide, …

Distribution Of Connectin (Titin) And Transverse Tubules At Myotendinous Junctions, Y. Shimada, F. Atsuta, M. Sonoda, M. Shiozaki, K. Maruyama

Scanning Microscopy

The ends of muscle fibers form many longitudinal projections which are further divided into numerous processes and attach to the collagen fibrils of tendons to form myotendinous junctions (MTJs). Immunocytochemical and electron microscopic observations on pectoralis muscles of the chicken revealed the presence of an elastic filamentous protein, connectin (titin), within the terminal sarcomere on the side adjacent to the terminal Z bands, and the absence of connectin and myosin and the presence of actin at the apical sarcoplasmic region of MTJ processes between the terminal Z band and the MTJ sarcolemma. Intermediate voltage electron microscopy showed that T tubules …

Preparation Of Cereals And Grain Products For Transmission Electron Microscopy, Donald B. Bechtel

Food Structure

This tutorial specifically addresses the techniques used in the processing of cereals and grain products for various aspects of transmission electron microscopy. Methods covered include sample treatment, chemical and physical fixation, dehydration, embedding, sectioning techniques, immunocytochemistry, enzymatic digestions, carbohydrate localization, and lectin binding. The primary goal is to provide information on the preparation of cereals and cereal-based products for microscopic analysis and to assist the reader in solving technical problems associated with studying cereals or other difficult-to-prepare samples.

Bioapplication Of Colloidal Gold In Microbiological Immunocytochemistry, Julian E. Beesley

Scanning Microscopy

Microbiological organisms are an ubiquitous group of animals encompassing bacteria, viruses, protozoa, algae and fungi. They are adapted for survival in many diverse habitats and exert a profound effect on man and his environment. Colloidal gold electron immunocytochemistry is a useful technique for studying these organisms and may be applied in several ways. The post-embedding technique is used to detect internal antigens, whilst the pre-embedding technique is employed for the detection of external antigens. In contrast the immuno-negative stain technique is applied to detect antigens on structures such as viruses or bacterial pili which may be dried down onto an …

Endothelialization Of A New Dacron Graft In An Experimental Model: Light Microscopy, Electron Microscopy And Immunocytochemistry, G. Pasquinelli, P. Preda, T. Curti, M. D'Addato, R. Laschi

Scanning Microscopy

Two types of synthetic vascular grafts, Dacron Triaxial and Dacron Gelseal Triaxial, were implanted into both the common carotids of sheep. The animals were sacrificed 1, 2, 8, and 16 weeks after surgery. Multiple specimens, obtained from grafts and anastomoses, were studied by light microscopy, transmission and scanning electron microscopy. A parallel immunocytochemical analysis was performed on some specimens. Dacron Triaxial grafts failed to develop a complete neointimal coverage. Myofibroblasts and fibroblasts were the dominant cells in such synthetic graft. Moreover, focal areas of stripping, platelet deposition, and thrombosis were observed at 8 and 16 weeks.

In contrast, a stable …

Early Cardiogenesis In The Newt Embryo, R. Hirakow, S. Komazaki, T. Hiruma

Scanning Microscopy

The migration of cardiogenic cells and the formation of a tubular heart in newt embryos were examined mainly by scanning electron microscopy (SEM). Cardiogenic cells are known to localize at the border region of lateral mesoderm migrating in the space between the ectoderm and the endoderm. They initially (before stage 20 or mid-neurula) appeared to attach to the basal surface of the ectoderm, whereas later (after stage 22 or late neurula) they changed their scaffold to the endoderm. On the scaffold cell surface, very fine fibrils of extracellular matrix (ECM) were found. These fibrils were proved to be composed partly …

Cryoultramicrotomy And Immunocytochemistry In The Analysis Of Muscle Fine Structure, L. -E. Thornell, G. S. Butler-Browne, E. Carlsson, H. M. Eppenberger, D. O. Fürst, B. K. Grove, B. Holmbom, J. V. Small

Scanning Electron Microscopy

Cryoultramicrotomy, which avoids the use of harsh fixation procedures, deleterious dehydration and plastic embedding can be combined with immunocytochemis try to determine the ultra-structural localization of cellular proteins. Our attempts to use the cryosectioning technique in combination with immunolabelling to bridge the gap between light and electron microscopic analysis of muscle morphology have enabled us to obtain new information on fibre typing at the ultrastructural level. Furthermore, we have obtained a marked improvement in the resolution of myofibrillar structures by using semithin cryosections for fluorescence microscopy. Data are also presented on correlated light and electron microscope immunocytochemistry of myocardial intermediate …

Scanning Electron Microscopical And Histochemical Study Of The Endoderm In The Early Chick Embryo, Marjorie A. England, Jennifer Wakely

Scanning Electron Microscopy

The endoderm of gastrulating chick embryos shows regional variations in cell shape and size. These were studied by scanning electron microscopy, histochemistry and immunofluorescence. Particular attention was given to the distribution of the cytoskeleton. Four zones of differing morphology were observed. The changing size and shape of these zones could be correlated with the entry of the definitive endoblast through the primitive streak, displacing existing primary hypoblast to the edges of the area pellucida. Endodermal cells were shown to have a well organised cytoskeleton. The cytoskeletons of individual cells were linked to give a cytoskeletal network extending across the endoderm …

Low Temperature Embedding, E. Carlemalm, W. Villiger, J. -D. Acetarin, E. Kellenberger

Scanning Electron Microscopy

The Lowicryl resins K4M and HM20 are methacrylate/acrylate based formulations which can re used for embedding biological material at low temperature in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. The resins are applicable over a very extended temperature range, approximately 210°K to 340°K. Even lower temperatures down to ca. 190°K can be reached with two new resins, K11M and HM23. Test embeddings of unfixed material after freeze-substitution have given promising results which could re useful for imnunocytochemical labeling. Lipid extraction is small or absent when the two new resins are used in combination with …

Immunocytochemical Labeling Of Enzymes In Low Temperature Embedded Plant Tissue: The Precursor Of Glyoxysomal Malate Dehydrogenase Is Located In The Cytosol Of Watermelon Cotyledon Cells, C. Sautter

Scanning Electron Microscopy

The Lowicryl-technique in combination with protein A gold was used in order to localize the precursor of glyoxysomal malate dehydrogenase in watermelon cotyledons. Preservation of the antigen was evaluated by a preembedding technique in isolated organelles. The glyoxysomal malate dehydrogenase was localized in tissue sections by a postembedding technique. Antigens of glyoxysomal malate dehydrogenase were found in the glyoxysomal matrix and in the cytosol, whereas the endoplasmic reticulum was completely free of labeling. Controls are presented by preimmunserum, by a serum against various proteins of the glyoxysomal membrane and by application of cycloheximide in order to inhibit translation at cytosolic …

To Resin Or Not To Resin In Immunocytochemistry, J. J. Wolosewick

Scanning Electron Microscopy

Hydrated resinless sections produced by a variety of methods (cryoultramicrotomy or polyethylene glycol techniques) appear to be excellent specimens for post-embedding immunocytochemistry at the electron microscopic level. A perplexing problem is the lack of apparent penetration throuqhout the section thickness of particulate probes such as ferritin or colloidal gold. This report draws attention to the possible causes for this phenomenon namely the size of the probe and the possible effects of fixation or processing. Smaller probes, gentler fixation or permeabilization procedures and increased incubation times all seem to be logical approaches to increasing penetration of immunoreagents into thick hydrated sections.