scholarly journals Ia antigens in plastic-embedded tissues: a post-embedding immunohistochemical study.

1986 ◽  
Vol 34 (6) ◽  
pp. 827-831 ◽  
Author(s):  
W Hermanns ◽  
F Colbatzky ◽  
A Günther ◽  
B Steiniger
Keyword(s):  
Rat Tissues ◽  
Thin Sections ◽  
Tissue Sections ◽  
Histocompatibility Complex ◽  
Embedding Technique ◽  
Ia Antigens ◽  
Distinct Cell ◽  
Plastic Embedding ◽  
Indirect Immunoperoxidase ◽  
Cryostat Sections

The aim of the present study was to establish a plastic embedding technique that makes possible the immunohistochemical demonstration of class II major histocompatibility complex (MHC) antigens (Ia antigens) in undecalcified joint tissues. Therefore a series of fixatives and dehydrating agents was tested for saving Ia immunoreactivity by post-embedding immunostaining of thin sections (2 microns) of rat tissues that had been embedded in glycol methacrylate (GMA), and by comparing with cryostat sections. An indirect immunoperoxidase and the avidin-biotin complex (ABC) technique were used. Combined with fixation by 4% formaldehyde, dehydration with GMA was found to give the best preservation of Ia antigenicity, followed by dehydration with ethylene glycol. The thinness of tissue sections facilitated the association of Ia antigens with different subcellular compartments in distinct cell populations. These various patterns are described.

scholarly journals Protein-embedding Technique: A Potential Approach to Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections

2005 ◽  
Vol 53 (9) ◽  
pp. 1167-1170 ◽  
Author(s):  
Shan-Rong Shi ◽  
Cheng Liu ◽  
Jeanette Perez ◽  
Clive R. Taylor
Keyword(s):  
Solid Phase ◽  
Antigen Retrieval ◽  
Polymer Microsphere ◽  
Thin Sections ◽  
Tissue Sections ◽  
Embedding Technique ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Two Phases ◽  
Formalin Fixed

A serial study was performed to develop a protein-embedding technique for standardization of immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) tissue sections. A protein carrier matrix must have two phases, a liquid phase to allow uniform mixing of a protein and a solid phase forming a ‘block’ that can be fixed and processed in the same manner as human tissue. This standardized protein block would serve as a source of thin sections for control of IHC and therefore must also withstand the boiling conditions of antigen retrieval (AR). After multiple experiments, a method was developed utilizing polymer microsphere (beads) as a support medium for protein. The method showed particular promise for quantitative IHC.

Ultrastructural localization of carbonic anhydrase in mouse gastric parietal cells

1978 ◽  
Vol 36 (2) ◽  
pp. 532-533
Author(s):  
N. Sugai ◽  
S. Ito
Keyword(s):  
Carbonic Anhydrase ◽  
Picric Acid ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Parietal Cells ◽  
Thin Sections ◽  
Tissue Sections ◽  
Gastric Parietal Cells ◽  
Cryostat Sections ◽  
Precise Distribution

The histochemical localization of carbonic anhydrase in parietal cells has been described by a number of investigators and its presence in these cells at the ultrastructural level has been also reported. However the precise distribution of this enzyme is not clear and there are some questions regarding the validity of the histochemical reaction. In the present study, various modifications of the technique were explored and it was found that tissues fixed in buffered formaldehyde, glutaraldehyde with trinitrocresol or picric acid retained good reaction product localization of this enzyme. Cryostat sections of the fixed tissues were treated with solutions recommended by Hannson. Incubation times that were most favorable 5 to 10 min for light microscopy and 8 to 15 min for electron microscopy. For ultrastructural observations of thin sections, it was found to be important that the reacted tissue sections were post osmicated with 1% osmium tetroxide in 1. 5% potassium ferrocyanide or with aqueous 1% 0s04 for only 3 to 5 min.

scholarly journals THE EFFECT OF COENZYME Q ON THE HISTOCHEMICAL SUCCINIC TETRAZOLIUM REDUCTASE REACTION: A HISTOCHEMICAL STUDY

1967 ◽  
Vol 15 (4) ◽  
pp. 216-224 ◽  
Author(s):  
CHARLES A. HORWITZ ◽  
LUIS BENITEZ ◽  
MARGARET BRAY
Keyword(s):  
Histochemical Study ◽  
Coenzyme Q ◽  
Succinic Dehydrogenase ◽  
Test System ◽  
Rat Tissues ◽  
Tissue Sections ◽  
Normal Rat ◽  
Electron Carriers ◽  
Cryostat Sections ◽  
Film Method

The role played by coenzyme Q (CoQ) in the succinic tetrazolium reductase reaction was investigated. Fresh cryostat sections of normal rat tissues were extracted with acetone to remove CoQ from the tissue sections and the acetone-extracted sections were reconstituted with CoQ-lecithin complexes. The incubation film method for tetrazolium reductases was used as a test system, using succinate as a substrate. Sections from which CoQ had been selectively extracted did not reduce the tetrazolium to its formazan, whereas those acetone-extracted sections that were treated with CoQ-lecithin complexes or with the electron carrier phenazine methosulfate reacted positively. It was concluded that the succinic tetrazolium reductase reaction requires intermediate electron carriers, mainly CoQ. It follows that nitro blue tetrazolium cannot accept electrons directly from the flavins. Therefore, the tetrazolium reductase reaction depends not only on the amount of succinic dehydrogenase and flavins but also on the amount of CoQ in the tissue.

High-resolution scanning electron microscopy (HRSEM) of mitochondria from various rat tissues

1988 ◽  
Vol 46 ◽  
pp. 14-15
Author(s):  
P.J. Lea ◽  
M.J. Hollenberg
Keyword(s):  
Electron Microscopy ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Rat Hepatocytes ◽  
Three Dimensions ◽  
Objective Lens ◽  
Rat Tissues ◽  
Thin Sections ◽  
Reconstruction Methods ◽  
Scanning Electron

Our current understanding of mitochondrial ultrastructure has been derived primarily from thin sections using transmission electron microscopy (TEM). This information has been extrapolated into three dimensions by artist's impressions (1) or serial sectioning techniques in combination with computer processing (2). The resolution of serial reconstruction methods is limited by section thickness whereas artist's impressions have obvious disadvantages.In contrast, the new techniques of HRSEM used in this study (3) offer the opportunity to view simultaneously both the internal and external structure of mitochondria directly in three dimensions and in detail.The tridimensional ultrastructure of mitochondria from rat hepatocytes, retinal (retinal pigment epithelium), renal (proximal convoluted tubule) and adrenal cortex cells were studied by HRSEM. The specimens were prepared by aldehyde-osmium fixation in combination with freeze cleavage followed by partial extraction of cytosol with a weak solution of osmium tetroxide (4). The specimens were examined with a Hitachi S-570 scanning electron microscope, resolution better than 30 nm, where the secondary electron detector is located in the column directly above the specimen inserted within the objective lens.

Flat embeddment of monolayer cells, tissue sections, and other cellular materials attached onto glass substrate

1990 ◽  
Vol 48 (3) ◽  
pp. 766-767
Author(s):  
Jeffrey P. Chang ◽  
Jaang J. Wang
Keyword(s):  
Present Report ◽  
Cellular Materials ◽  
Cover Glass ◽  
Tissue Sections ◽  
Embedding Technique ◽  
Exfoliated Cells ◽  
Black Paper ◽  
Cell Concentrations ◽  
Flat Embedding

Flat embeddment of certain specimens for electron microscopy is necessary for three classes of biological materials: namely monolayer cells, tissue sections of paraffin or plastics, as well as cell concentrations, exfoliated cells, and cell smears. The present report concerns a flat-embedding technique which can be applied to all these three classes of materials and which is a modified and improved version of Chang's original methodology.Preparation of coverglasses and microslides. Chemically cleaned coverglasses, 11 × 22 mm or other sizes, are laid in rows on black paper. Ink-mark one coner for identifying the spray-side of the glass for growing cells. Lightly spray with Teflon monomer (Heddy/Contact Inductries, Paterson, NO 07524, U.S.A.) from a pressurized can. Bake the sprayed glasses at 500°F for 45 min on Cover-Glass Ceramic Racks (A. Thomas Co. Philadelphia), for Teflon to polymerize.Monolayer Cells. After sterilization, the Teflon-treated coverglasses, with cells attached, are treated or fixed in situ in Columbia staining dishes (A. Thomas Co., Philadelphia) for subsequent processing.

scholarly journals Accessory and stimulating properties of dendritic cells and macrophages isolated from various rat tissues

1982 ◽  
Vol 156 (1) ◽  
pp. 1-19 ◽  
Author(s):  
WEF Klinkert ◽  
JH LaBadie ◽  
WE Bowers
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cell Activity ◽  
Accessory Cell ◽  
Nonspecific Esterase ◽  
Rat Tissues ◽  
Low Density ◽  
Γ Irradiation ◽  
Accessory Cells ◽  
Ia Antigens

Single cell suspensions of rat lymphoid and nonlymphoid tissues were fractionated on discontinuous gradients of bovine serum albumin into high density and low density subfractions. In general, accessory activity required for responses of periodate-treated T lymphocytes was recovered only in a low density population containing a small percent of the total fractionated cells from lymph nodes, spleen, liver, skin, and peritoneal exudates. Further purification always led to an increase of both accessory activity and number of dendritic cells present in nonrosetting and nonadherent populations. After purification, a high recovery of the total accessory activity was found in fractions that contained a high percentage of dendritic cells resulting in a more than 1,000-fold enrichment in accessory activity per cell. No other fraction obtained during the purification contained significant accessory activity. In all cases, macrophage-enriched populations lacked accessory cell activity. With the exception of peritoneal exudate cell preparations, which contained an inhibitory cell, the level of accessory activity in a given population was always found to be a function of the number of dendritic cells present. Dendritic cells from all sources were nonadherent, nonphagocytic, radio- resistant, and nonspecific esterase negative. They expressed Ia antigens and lacked Fc receptors. Both epidermal and lymph node dendritic cells contain Birbeck granules, subcellular structures previously described only for Langerhans cells. Accessory activity requires viable dendritic cells but is unaffected by 1,000 rad of γ-irradiation. However, ultraviolet irradiation abolished the activity of accessory cells. The cells that responded to periodate were IgG-negative T cells, whereas IgG-positive B cells could not be stimulated under the same conditions. Only periodate-treated T cells and dendritic cells were needed for responses to occur; removal of virtually all macrophages from these purified preparations had no effect. Dendritic cells were also required as stimulators in mixed leukocyte cultures, whereas macrophages, even though Ia positive, were inert.

Use of Plastic Embedding Materials

1974 ◽  
Vol 1 (4) ◽  
pp. 147-148 ◽  
Author(s):  
J. A. Sandham ◽  
J. D. McEwen
Keyword(s):  
Clinical Practice ◽  
Embedding Technique ◽  
Plastic Embedding

The use of a plastic embedding technique has been described and its relevance in orthodontic teaching and clinical practice discussed. We have found that many aspects of orthodontic instruction benefit from the use of these materials.

scholarly journals Evaluation of silicon and germanium retention in rat tissues and diatoms during cell and organelle preparation for electron probe microanalysis.

1975 ◽  
Vol 23 (5) ◽  
pp. 348-358 ◽  
Author(s):  
C W Mehard ◽  
B E Volcani
Keyword(s):  
Rat Liver ◽  
Freeze Substitution ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Rat Tissues ◽  
Cell Organelles ◽  
Thin Sections ◽  
X Ray ◽  
Low Viscosity ◽  
Silicon And Germanium

Chemical, radiochemical and x-ray microanalysis assays were used to define parameters of silicon (Si) retention during preparation og biologic samples (rat liver, spleen, kidney, lung, diatoms and cell organelles) for x-ray microanalysis, Due to its longer half-life 68-Fe was used in some cases to trace SI. Leaching of Si from cells and organelles by the aqueous preparation media was overcome by use of the freeze-substitution process. Cells were treated with 30% glycerol hypertonic sucrose medium to reduce ice damage. Embedment in Spurr's low viscosity epoxy resin medium caused no apparent Si loss. A semiquantitative evaluation showed 0.5 x 10-8 to 0.3 x 10-17 g detectable Si in isolated rat liver mitochondria in thin sections, which is within the instrument's range of detection. This study indicateds that the presence of Si in the mitochondria is not the rsult of contamination.

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