DNA Fragmentation Immunohistochemistry Kit

Sample

Paraffin-embedded tissue sections, frozen tissue sections, or single cell suspensions fixed on slides.

Species Reactivity

N/A

Detection Method

Immunohistochemistry Kit

Intended Use

Our DNA Fragmentation Detection Kit exploits the fact that apoptotic endonucleases not only affect cellular DNA by producing the classical DNA ladder but also generate free 3'-OH groups at the ends of these DNA fragments. These free 3'-OH groups are end-labeled by the DNA Fragmentation Detection Kit allowing for the detection of apoptotic cells using a molecular biology-based, end-labeling technique.

Contents of Kit

The DNA Fragmentation Detection kit supplies sufficient reagents to specimens of approximate size 2.5 cm<sup>2.
1. Proteinase K, a pH-stabilized Solution
2. TdT Equilibration Buffer
3. TdT Labeling Reaction Mix, an optimized mix of labeled and unlabeled nucleotides
4. TdT Enzyme, Terminal Deoxynucleotidyl Transferase.
5. Stop Buffer
6. Block Buffer
7. 25× Streptavidin-HRP Conjugate
8. DAB Concentrate
9. DAB Reaction Buffer
10. Methyl Green Counterstain</sup>

Storage

The DNA Fragmentation Detection Kit components are shipped on cold pack. Upon receipt, store kit at -20°C in a non-frost-free freezer. For long term storage, it is recommended that you aliquot and freeze the TdT Enzyme (Component 4), TdT Labeling Reaction Mix (Component 3), 25× Streptavidin-HRP Conjugate (Component 7), and Proteinase K (Component 1) at -20 °C. Thirty (30) minutes prior to use of each component, thaw component and keep on cold block or on ice. Return the components to -20°C for long term storage or 4-8°C for short term storage (up to 2 weeks) immediately after use. Special care should be taken to keep TdT Enzyme (Component 4), TdT Labeling Reaction Mix (Component 3), 25x Streptavidin-HRP Conjugate (Component 7), and Proteinase K (Component 1) cold by pulling out the number of aliquots needed for the test, keeping them on ice, and leaving the remaining aliquots at -20°C.
All of the reagents included with the DNA Fragmentation Detection Kit have been tested for stability. Reagents should not be used beyond the stated expiration date.

General Description

Cell death occurs by two major mechanisms, necrosis and apoptosis. Apoptosis is also known as programmed cell death or ankoikis (a form of apoptosis which is induced by anchorage-dependent cells detaching from the surrounding extracellular matrix). Apoptosis leads to the elimination of cells without releasing harmful substances into the surrounding area. Too little or too much apoptosis plays a role in a great many diseases.
When apoptosis functions inappropriately, cells that should be eliminated survive and potentially become immortal, as in cancer or leukemia. When apoptosis works overly well, too many cells may 'die' and the result may be grave tissue damage. This is the case in stroke and neurodegenerative disorders such as Alzheimer, Huntington and Parkinson diseases. The term 'apoptosis' refers only to the structural changes a cell goes through during the process of programmed cell death and not to the process itself. Classical necrotic cell death occurs due to noxious injury or trauma to the cell while apoptosis is an energy dependent mechanism that takes place during normal cell development. While necrotic cell death results in cell lysis, cellular apoptosis is characterized morphologically by cell shrinkage, nuclear pyknosis, chromatin condensation, and blebbing of the plasma membrane. Apoptosis is the result of a cascade of molecular and biochemical events involving endogenous endonucleases that cleave DNA into the prototypical 'ladder of DNA fragments' that may be visualized in agarose gels. Observation of oligonucleosomal DNA fragments by DNA laddering has long been the most acceptable and only available assay for the detection of apoptosis.