scholarly journals Developmental changes in heparan sulfate expression: in situ detection with mAbs.

1992 ◽  
Vol 119 (4) ◽  
pp. 961-975 ◽  
Author(s):  
G David ◽  
X M Bai ◽  
B Van der Schueren ◽  
J J Cassiman ◽  
H Van den Berghe
Keyword(s):  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Regional Differences ◽  
Cell Types ◽  
Basement Membranes ◽  
Developmentally Regulated ◽  
Tissue Sections ◽  
Heparan Sulfates ◽  
In Situ Detection

Two mAbs that are specific for heparan sulfate-related epitopes have been raised and used to analyze the cellular and tissular distribution of this glycosaminoglycan during development. mAb 10E4 reacts with an epitope that occurs in native heparan sulfate chains and that is destroyed by N-desulfation of the glycosaminoglycan. The antibody does not react with hyaluronate, chondroitin sulfate, or DNA, and reacts only poorly with heparin. The reactivity of proteoglycan extracts or tissue sections with the 10E4 antibody is completely abolished by heparitinase, but is only partially affected by heparinase. mAb 3G10, in contrast, reacts only with heparitinase-treated heparan sulfate chains, proteoglycans, or tissue sections. The 3G10 epitope is destroyed by treatment with mercuric acetate, which indicates that the desaturated uronate generated by the lyase is essential for the reactivity of the antibody. The 3G10 epitope is not generated by treating heparan sulfate proteoglycans with heparinase or chondroitin sulfate proteoglycans with chondroitin sulfate lyases, which indicates that the 3G10 antibody recognizes desaturated uronates that occur in specific structural contexts. The antibody 10E4 and, after heparitinase treatment, the antibody 3G10 decorate the surfaces of many cell types and the extracellular matrix in proximity of the cells, in particular, the basement membranes. The analysis of embryonic and adult tissues reveals important temporal and regional differences in the abundance of the 10E4 and 3G10 epitopes at these sites. Moreover, the staining pattern of the two antibodies is not always superimposable, which is indicative of regional differences in the exposure or structure of the tissular heparan sulfates. As a whole the results suggest that heparan sulfate abounds at sites of active morphogenesis and that the expression of this glycosaminoglycan is developmentally regulated.

Porcine Repeat Element DNA: In Situ Detection of Xenotransplanted Cells

1995 ◽  
Vol 4 (2) ◽  
pp. 253-256 ◽  
Author(s):  
Henry F. Oettinger ◽  
Amelie Rodrigue-Way ◽  
Joyce J. Bousquet ◽  
Albert S.B. Edge
Keyword(s):  
Southern Blotting ◽  
Host Tissue ◽  
Repeat Element ◽  
Tissue Analysis ◽  
Specific Staining ◽  
Tetrazolium Chloride ◽  
Tissue Sections ◽  
Blue Tetrazolium Chloride ◽  
In Situ Detection

Using a digoxygenin-labelled DNA probe derived from the porcine repeat element PRE-1, we have developed a protocol for the detection of transplanted porcine islets and hepatocytes against a background of murine host tissue. Analysis of this probe by Southern blotting indicated that PRE-1 hybridizes to pig genomic DNA but not to human or mouse DNA. On tissue sections, hybridizing probe was detected using alkaline phosphatase-conjugated anti-digoxygenin antibody visualized with 5-bromo-4-chloro-3-indolyl-phosphate/4-nitro-blue tetrazolium chloride (BCIP/ NBT) substrate. We have demonstrated sensitive and highly specific staining of porcine nuclei in fixed, paraffin embedded tissue sections, and have applied the technique to detect porcine pancreatic islets and hepatocytes transplanted into murine kidney and spleen. Applications of this technique include detection of transplanted cells or organs across a variety of xenogeneic barriers.

scholarly journals Spatial charting of single cell transcriptomes in tissues

2021 ◽  
Author(s):  
Nicholas Navin ◽  
Runmin Wei ◽  
Siyuan He ◽  
Shanshan Bai ◽  
Emi Sei ◽  
...  
Keyword(s):  
Single Cell ◽  
Spatial Organization ◽  
Spatial Information ◽  
Ductal Carcinoma ◽  
Single Cells ◽  
Cell Types ◽  
Spatial Structures ◽  
Tissue Sections ◽  
Normal Mouse Brain

Single cell RNA sequencing (scRNA-seq) methods can profile the transcriptomes of single cells but cannot preserve spatial information. Conversely, spatial transcriptomics (ST) assays can profile spatial regions in tissue sections, but do not have single cell genomic resolution. Here, we developed a computational approach called SChart, that combines these two datasets to achieve single cell spatial mapping of cell types, cell states and continuous phenotypes. We applied SChart to reconstruct cellular spatial structures in existing datasets from normal mouse brain and kidney tissues to validate our approach. We also performed scRNA-seq and ST experiments on two ductal carcinoma in situ (DCIS) tissues and applied SChart to identify subclones that were restricted to different ducts, and specific T cell states adjacent to the tumor areas. Our data shows that SChart can accurately map single cells in diverse tissue types to resolve their spatial organization into cellular neighborhoods and tissue structures.

Phosphotungstic Acid As An Electron Stain

1968 ◽  
Vol 26 ◽  
pp. 36-37
Author(s):  
Daniel C. Pease
Keyword(s):  
Chondroitin Sulfate ◽  
Room Temperature ◽  
Phosphotungstic Acid ◽  
Freeze Substitution ◽  
Basement Membranes ◽  
Effective Electron ◽  
Tissue Sections ◽  
Brush Borders ◽  
Effects Of Ph ◽  
Acid Environment

Under certain conditions phosphotungstic acid (PTA) can be used as an effective electron stain for demonstrating polysaccharide moieties in tissue sections. There it selectively combines with the glycoproteins of basement membranes, the mucoid layers associated with brush borders, the mucus of goblet cells, the chondroitin sulfate of cartilage matrix, glyco-gen, and “glycocalyces” in general. The initial published investigation indicated that an acid environment was essential for this specificity, but the effect of pH was not systematically explored.Rib cartilage of newborn rats was chosen for a detailed study of the effects of pH on PTA staining, particularly to provide ancillary information on the formation, secretion and deposition of chondroitin sulfate. Cartilage was prepared without chemical fixation by “freeze-substitution” with 70% glycol, or by “inert dehydration” with glycol at room temperature. Dehydrated material was embedded in prepolymerized hydroxy-propyl methacrylate without an intermediate solvent.

scholarly journals In situ ultrastructural characterization of chondroitin sulfate proteoglycans in normal rabbit aorta.

1992 ◽  
Vol 40 (2) ◽  
pp. 251-263 ◽  
Author(s):  
Z S Galis ◽  
M Z Alavi ◽  
S Moore
Keyword(s):  
Chondroitin Sulfate ◽  
Rabbit Aorta ◽  
Specific Information ◽  
In Situ Characterization ◽  
Tissue Sections ◽  
Ultrastructural Characterization ◽  
The Media ◽  
Very High

We used a monoclonal antibody recognizing chondroitin sulfate (CS) to investigate by immunocytochemistry the characteristics displayed in situ by aortic proteoglycans (PG) containing CS side chains. The antibody specifically precipitated metabolically labeled PG from aortic extracts. Anti-CS specificity was also tested directly on tissue sections and was confirmed by the virtual abolition of immunolabeling on those previously digested with CS-specific enzymes. The overall CS-PG distribution assessed by light microscopy after embedding in Lowicryl KM4 by silver-enhanced immunogold recapitulated that obtained on frozen sections with immunoperoxidase. Extracellular concentrations of CS-PG were very high in the innermost regions of aorta and decreased in the media. The reaction was weak and diffuse in the adventitia. By electron microscopy, the detailed labeling of CS-PG discriminated patterns of organization at both the regional and the molecular level and enabled morphometric estimations. In relation to other components of the extracellular matrix, we found that CS-PG and elastin mutually excluded each other, while two types of CS-PG were differently associated with collagen within media or adventitia. The use of high-resolution immunodetection for the in situ characterization of aortic CS-PG could add specific information relevant to many biological processes in which these molecules have been implicated.

In situ PCR-associated immunohistochemistry identifies cell types harbouring the Maedi-Visna virus genome in tissue sections of sheep infected naturally

2003 ◽  
Vol 107 (2) ◽  
pp. 121-127 ◽  
Author(s):  
Maria Luisa Carrozza ◽  
Maurizio Mazzei ◽  
Patrizia Bandecchi ◽  
Mario Arispici ◽  
Francesco Tolari
Keyword(s):  
Cell Types ◽  
Virus Genome ◽  
In Situ Pcr ◽  
Tissue Sections ◽  
Visna Virus ◽  
Maedi Visna Virus

scholarly journals Thermal stability of the helical structure of type IV collagen within basement membranes in situ: determination with a conformation-dependent monoclonal antibody.

1984 ◽  
Vol 99 (4) ◽  
pp. 1405-1409 ◽  
Author(s):  
T F Linsenmayer ◽  
E Gibney ◽  
J M Fitch ◽  
J Gross ◽  
R Mayne
Keyword(s):  
Thermal Stability ◽  
Type Iv Collagen ◽  
Helical Structure ◽  
Basement Membranes ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tissue Sections ◽  
Type Iv ◽  
The Stability ◽  
Thermal Stability Of

To examine the thermal stability of the helical structure of type IV collagen within basement membranes in situ, we have employed indirect immunofluorescence histochemistry performed at progressively higher temperatures using a conformation-dependent antibody, IV-IA8. We previously observed by competition enzyme-linked immunosorbent assay that, in neutral solution, the helical epitope to which this antibody binds undergoes thermal denaturation over the range of 37-40 degrees C. In the present study, we have reacted unfixed cryostat tissue sections with this antibody at successively higher temperatures. We have operationally defined denaturation as the point at which type IV-specific fluorescence is no longer detectable. Under these conditions, the in situ denaturation temperature of this epitope in most basement membranes is 50-55 degrees C. In capillaries and some other small blood vessels the fluorescent signal is still clearly detectable at 60 degrees C, the highest temperature at which we can confidently use this technique. We conclude that the stability of the helical structure of type IV collagen within a basement membrane is considerably greater than it is in solution, and that conformation-dependent monoclonal antibodies can be useful probes for investigations of molecular structure in situ.

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