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Detection of apoptotic and phagocytic cells in paraffin-embedded rat mammary gland tissue sections using ApopTag ISOL Dual Fluorescence Apoptosis Dete...
ApopTag Dual ISOL Chemistry
ApopTag® ISOL Dual Fluorescence Apoptosis Detection Kit (DNase Types I & II)
Description:
ApopTag® ISOL Dual Fluorescence Apoptosis Detection Kit (DNase Types I & II)
Trade Name:
Chemicon (Millipore)
Qty/Pk:
25 assays
Product Overview:
The ApopTag ISOL Dual Fluorescence Kit utilizes a proprietary double hairpin, dual fluorescently labeled oligonucleotide labeling process to detect and distinguish between typical apoptotic DNA breaks induced by either DNase I or DNase II enzyme activities. The Vaccinia Topoisomerase I mediated ligation reaction is adaptable for staining paraffin-embedded tissue, frozen tissue sections, cell suspensions, and adherent cells. In situ staining for DNA fragmentation (as in the ISOL method) is both a means of detection for rare cells and an analytical test of those cells' DNA. ApopTag ISOL Kits facilitate the differentiation of apoptotic cells from necrotic or transiently damaged cells. While conventional in situ detection techniques such as ISEL (Klenow DNA polymerase), TUNEL (terminal deoxynucleotidyl transferase, TdT) and ISNT (DNA Polymerase I) are useful in detecting internucleosomal DNA cleavage, they do not differentiate DNase Type I and DNase Type II cleavage which results from the activation of apoptotic endonucleases. When ISOL was used for direct comparison with TUNEL in specimens without necrosis, the results have been concordant. In specimens presenting necrosis, the better selectivity of ISOL was proven.
Background Information:
The ApopTag ISOL Fluorescence Apoptosis Detection (DNase Types I & II) technique is based upon the biochemical specificity of the enzymes T4 DNA ligase and Vaccinia Virus DNA Topoisomerase I and the unique, dual labeled, dual hairpin oligonucleotide (8,9,10). This self-annealing oligo contains two sets of complementary base sequences that spontaneously form two duplex segments resulting in a dual hairpin secondary structure (Figure 1). The oligo also contains two internal fluorescent labels which are located at opposite poles of the dual hairpin structure. At one pole is a FAM internal label and the other pole contains a CR590 label. The basis of the detection mechanism relies on the 5'-CCCTT-3' Topoisomerase I recognition site located in the middle of the dual hairpin structure. The Topoisomerase I cuts the DNA at the 3' end of the recognition site causing the dual hairpin oligo to dissociate into two separate differentially labeled hairpin oligonucleotides. The Toposiomerase I remains covalently bound to the oligo containing the recognition site and the FAM label while the other CR590 containing oligo dissociates. Hence, two differentially labeled hairpin oligo probes are created. The biochemical specificity of the provided enzymes impacts the detection aspect of the protocol, in that Vaccinia Topoisomerase I will recognize and ligate the FAM oligo to 5'-OH 3'-PO4 groups (DNase type II specific cut) whereas T4 DNA Ligase will recognize and ligate the CR590 labeled oligo to 5'-PO4 3'-OH groups (DNase type I specific cut). The ISOL Kit does not label nicks, gaps, single-stranded DNA, 3'-recessed ends or 3'-overhanging ends.
Species Reactivity:
All
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Components:
- T4 DNA Ligase Enzyme (Part. No.2007460) - 100 μL
- Dual Reaction Buffer (2X) (Part. No.2007464) - 375 μL
- Dual Labeled Oligo (Part. No.2007463) - 25 μL
- Vaccinia Topoisomerase I (Part. No. 2007459) - 250 μL
- Plastic Coverslips (Part. No. 90421) - 100 each
- Positive Control Slides (Part. No. 90422) - 2 each
- Proteinase K (Part. No. 90435) - 25 mg
Brand Family:
Chemicon
Storage Conditions:
1 year at -20°C from date of shipment
Product Name:
ApopTag® ISOL Dual Fluorescence Apoptosis Detection Kit (DNase Types I & II)