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Products >  Cell_Biology >  Apoptosis_Kits_and_Reagents >  In_situ_Apoptosis_Detection_Set

Apoptosis Assay: In situ Apoptosis Detection Set

During apoptosis, DNA is fragmented following the apoptotic activation of intracellular endonucleases. The In Situ Apoptosis Detection Set is designed to detect fragmented DNA histochemically by TUNEL (TdT-mediated dUTP Nick End Labeling). Via this method, fluorescein-labeled nucleotides are incorporated in situ onto the 3' ends of DNA fragments, allowing histologic localization and detection of individual apoptotic cells.

Apoptosis plays a significant role in various stages of cancer as well as cell development, growth and proliferation. Physical (e.g., radiation, heat) and chemical (e.g., medicine) factors can also induce the apoptotic state. One of the features of apoptotic cells is the cleavage of nuclear DNA to approximately 185 bp fragments. This fragmented DNA can be detected histochemically by the TUNEL method.

Principle

TUNEL, an acronym for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling, is an effective method for measuring DNA fragments resulting from the apoptotic activation of intracellular endonucleases. The TdT enzyme is used to label the 3'-OH ends of DNA fragments with fluorescein-dUTP. These fluorescence-labeled nucleotides are incorporated in situ onto DNA fragment ends, allowing histologic localization of individual cells. As a result, cells undergoing apoptosis can be easily detected by fluorescence microscopy. Since incorporated fluorescein-dUTP can also be detected using peroxidase-labeled anti-fluorescein antibody, it is possible to detect apoptotic cells using light microscopy.

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Features

  • Quick and easy detection of apoptotic cells
  • Sensitive detection of single cells in the initial stages of apoptosis
  • Specific staining of apoptotic and not necrotic cells
  • Flexible enough to be used with tissue section or fixed cell samples
  • Applicable to either fluorescence or light microscopy
  • Individual components are available separately
  • Control slide supplied for accuracy 
  • Non-radioactive

Applications

  • Fragmented DNA Detection by the TUNEL Method
  • Detection of DNA strand breaks in apoptotic cells via fluorescence or light microscopy

Components

MK500

1. Labeling Safe Buffer2 x 500 mL
2. TdT (terminal deoxynucleotidyl transferase) enzyme*2 x 50 mL
3. Anti-FITC HRP Conjugate1.5 mL
4. Control slides**2 slides
5. Permeabilization buffer2 x 1.0 mL

*Source: Recombinant E. coli encoding the TdT gene

**Control slides contain paraffin-embedded tissue sections of rat mammary gland. When used as a positive control, deparaffinization is required. Following deparaffinization and treatment with proteinase K, please follow the listed detection method protocol.

Storage

Shipped at –20°C.

Components should be stored separately as follows: Labeling Safe Buffer, TdT enzyme, and Permeabilization buffer at –20°C; Anti-FITC HRP Conjugate at 4°C* and Control slides at room temperature.**

*Store at 4°C when thawed.

**Store at room temperature after delivery; kit is shipped at -20°C.

Fragmented DNA Detection by the TUNEL Method 

  • Type: Direct TUNEL Labeling Assay
  • Used for: Detection of DNA strand breaks in apoptotic cells by flow cytometry or fluorescence microscopy.
  • Used to Assay: Cells in suspension (permanent cell lines, normal and tumor cells ex vivo), adherent cells, cytospins, cell smears, frozen or paraffin-embedded tissue sections.
  • Technique: End-labeling of DNA with fluorescein-dUTP, followed by direct analysis of fluorescent cells.
  • Time Required: 1–2 hrs. (Not including sample preparation, permeabilization, etc.)
  • Detection Method: Fluorescence and light microscopy
  • Number of Assays per Kit: 20



 
 
Products
Offers Cat. # Product Contents Size Price Units Select
MK503 Anti-FITC HRP Conjugate 20 Rxns $196.00
MK504 Control Slides 5 x 2 Slides $94.00
MK500 In Situ Apoptosis Detection Kit 20 Assays $214.00
MK501 Labeling Safe Buffer 20 Rxns $466.00
MK505 Permeabilisation Buffer 20 Rxns $80.00
MK502 TdT Enzyme 1 ug $214.00
 

 

 
 

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