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 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.

Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections

2007 ◽  
Vol 2 (6) ◽  
pp. 1508-1514 ◽  
Author(s):  
Gregor Obernosterer ◽  
Javier Martinez ◽  
Mattias Alenius
Keyword(s):  
Nucleic Acid ◽  
Locked Nucleic Acid ◽  
Mouse Tissue ◽  
Tissue Sections ◽  
In Situ Detection

scholarly journals Simultaneous in situ detection of mRNA and apoptotic cells by combined hybridization and TUNEL.

1996 ◽  
Vol 44 (12) ◽  
pp. 1497-1499 ◽  
Author(s):  
J Sträter ◽  
H Walczak ◽  
P H Krammer ◽  
P Möller
Keyword(s):  
Fas Ligand ◽  
Apoptotic Cells ◽  
Thymic Tissue ◽  
Double Labeling ◽  
Cytoplasmic Staining ◽  
Tissue Sections ◽  
In Situ Detection ◽  
Streptavidin Peroxidase ◽  
Apoptosis Related Gene

We established a new method to allow simultaneous in situ detection of mRNA expression and apoptotic DNA fragmentation in paraffin-embedded tissue sections. We used human thymic tissue to perform in situ hybridization with a digoxigenin-labeled CD95 (APO-1/Fas) ligand (CD95L)-specific probe followed by TdT-mediated biotin dUTP nick end-labeling (TUNEL) of apoptotic DNA fragments. Bound probes were visualized by an immunogold-silver enhancement technique and fragmented DNA was detected with a streptavidin-peroxidase system. This double labeling technique produced a distinct, dark cytoplasmic staining of CD95L mRNA-expressing cells and an intense red nuclear precipitate in apoptotic cells or bodies. This technique will be a useful tool for microtopographical analysis of apoptosis-related gene expression.

In situ detection of human Ig light-chain mRNA on formalin-fixed and paraffin-embedded tissue sections using digoxigenin-labelled RNA probes

1993 ◽  
Vol 25 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Langxing Pan ◽  
Lisa C. Happerfield ◽  
Lynda G. Bobrow ◽  
Peter G. Isaacson
Keyword(s):  
Light Chain ◽  
Tissue Sections ◽  
Rna Probes ◽  
In Situ Detection ◽  
Formalin Fixed

scholarly journals NOT ALL ANTIBODIES HAVE BEEN CREATED EQUAL: Antibodies validated for routinely processed tissue seldom apply to frozen tissue sections

2020 ◽  
Author(s):  
Maddalena M Bolognesi ◽  
Francesco Mascadri ◽  
Roberto Perego ◽  
Silvia Bombelli ◽  
Francesca M Bosisio ◽  
...  
Keyword(s):  
Frozen Sections ◽  
Specific Staining ◽  
Tissue Sections ◽  
Antibody Staining ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Data Validity ◽  
Context Dependent ◽  
Formalin Fixed

ABSTRACTA customized context-dependent validation of antibodies for the prospective use, rather than a general stamp of validity, is required for reproducibility and data validity, besides the need of standardized reagents. In-situ antibody staining is a technique broadly used in experimental settings and human diagnostic practice. The first typically, but not exclusively uses lightly fixed material (cell smears, frozen sections), the second, routinely processed formalin-fixed, paraffin-embedded (FFPE) tissue. Differently from techniques based on tissue extraction, there is little awareness of the context-dependent constraints inherent with either type of in situ staining except that antigen masking associated with routine tissue processing limits the range of useful antibodies. We applied a panel of 126 antibodies validated for FFPE to lightly fixed (acetone, formalin) frozen sections and found that less than 30% performed conservatively with all fixations, 35% preferred one fixation over another, 13% gave non-specific staining, 23% did not stain at all. Individual antibody variegation of the paratope fitness for the differentially fixed antigen may be the cause. Re-validation of established antibody panels is required when applied to sections whose fixation and processing is different from the tissue where they have been initially validated.

scholarly journals In situ detection of Ha-ras and c-myc mRNA in cancer cell lines and tissue sections of colorectal cancer using sulfonated DNA probes.

1988 ◽  
Vol 21 (2) ◽  
pp. 201-210 ◽  
Author(s):  
TAKUSHI MONDEN ◽  
HIDEKI MORIMOTO ◽  
MASAHIKO HIGASHIYAMA ◽  
MASAHIRO MUROTANI ◽  
NAOHIRO TOMITA ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Dna Probes ◽  
Tissue Sections ◽  
In Situ Detection
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