APOPTAG PEROXIDASE DET. KIT

Stock Code: 3589086
Manufacturer Part No: S7100

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Descriptions

Components

Equilibration Buffer: 3.0 mL -15°C to -25°CReaction Buffer 2.0 mL -15°C to -25°CTdT Enzyme 0.64 mL -15°C to -25°CStop/Wash Buffer 20 mL -15°C to -25°CAnti-Digoxigenin-Peroxidase* 3.0 mL 2°C to 8°CPlastic Coverslips 100 ea. Room Temp.Note: Separate purchase of DAB (Peroxidase Substrate) is required. It is not supplied with this kit.Number of samples per kit: Sufficient materials are provided to stain 40 tissue specimens of approximately 5 mm² each when used according to instructions. Reaction Buffer will be fully consumed before other reagents when kits are used for slide-mounted specimens.

General description

The ApopTag™ Peroxidase In Situ Apoptosis Detection Kit detects apoptotic cells in situ by labeling and detecting DNA strand breaks by the TUNEL method. The kit provides sufficient reagents for immunoperoxidase staining of 40 samples. Results are visualized using brightfield microscopy.

Apoptosis is a form of cell death that eliminates compromised or superfluous cells. It is controlled by multiple signaling and effector pathways that mediate active responses to external growth, survival, or death factors. Cell cycle checkpoint controls are linked to apoptotic enzyme cascades, and the integrity of these and other links can be genetically compromised in many diseases, such as cancer. There are many books in print and hundreds of recent review articles about all aspects of apoptosis (e.g. 7, 11, 19, 24, 39, 42) and the methods for detecting it (e.g. 10, 32, 36).

Of all the aspects of apoptosis, the defining characteristic is a complete change in cellular morphology. As observed by electron microscopy, the cell undergoes shrinkage, chromatin margination, membrane blebbing, nuclear condensation and then segmentation, and division into apoptotic bodies which may be phagocytosed (11, 19, 24). The characteristic apoptotic bodies are short-lived and minute, and can resemble other cellular constituents when viewed by brightfield microscopy. DNA fragmentation in apoptotic cells is followed by cell death and removal from the tissue, usually within several hours (7). A rate of tissue regression as rapid as 25% per day can result from apparent apoptosis in only 2-3% of the cells at any one time (6). Thus, the quantitative measurement of an apoptotic index by morphology alone can be difficult.

DNA fragmentation is usually associated with ultrastructural changes in cellular morphology in apoptosis (26, 38). In a number of well-researched model systems, large fragments of 300 kb and 50 kb are first produced by endonucleolytic degradation of higher-order chromatin structural organization. These large DNA fragments are visible on pulsed-field electrophoresis gels (5, 43, 44). In most models, the activation of Ca²⁺- and Mg²⁺-dependent endonuclease activity further shortens the fragments by cleaving the DNA at linker sites between nucleosomes (3). The ultimate DNA fragments are multimers of about 180 bp nucleosomal units. These multimers appear as the familiar "DNA ladder" seen on standard agarose electrophoresis gels of DNA extracted from many kinds of apoptotic cells (e.g. 3, 7,13, 35, 44).

Another method for examining apoptosis via DNA fragmentation is by the TUNEL assay, (13) which is the basis of ApopTag™ technology