Apoptosis Detection Using ApopTag Peroxidase In Situ Apoptosis Detection Kit from Chemicon (S7100)


Apoptosis Detection/TUNEL Staining Service

Description: ApopTag® In Situ Apoptosis Detection Kits label apoptotic cells in research samples by modifying DNA fragments utilizing terminal deoxynucleotidyl transferase (TdT) for detection of apoptotic cells by specific staining. The Kit has been qualified for use in histochemical and cytochemical staining of the following specimens: formalin-fixed, paraffin-embedded tissues, cryostat sections, cell suspensions, cytospins, and cell cultures. Whole mount-methods have been developed.

Principles of the Procedure: The reagents provided in ApopTag® Peroxidase Kits are designed to label the free 3'OH DNA termini in situ with chemically labeled and unlabeled nucleotides. The nucleotides contained in the Reaction Buffer are enzymatically added to the DNA by terminal deoxynucleotidyl transferase (TdT). TdT catalyzes a template-independent addition of nucleotide triphosphates to the 3'-OH ends of double-stranded or single-stranded DNA. The incorporated nucleotides form an oligomer composed of digoxigenin-conjugated nucleotide and unlabeled nucleotide in a random sequence. The ratio of labeled to unlabeled nucleotide in ApopTag® Peroxidase Kits is optimized to promote anti-digoxigenin antibody binding. The exact length of the oligomer added has not been measured.

DNA fragments which have been labeled with the digoxigenin-nucleotide are then allowed to bind an anti-digoxigenin antibody that is conjugated to a peroxidase reporter molecule. The bound peroxidase antibody conjugate enzymatically generates a permanent, intense, localized stain from chromogenic substrates, providing sensitive detection in immunohistochemistry or immunocytochemistry (i.e. on tissue or cells). This mixed molecular biological-histochemical systems allows for sensitive and specific staining of very high concentrations of 3'-OH ends that are localized in apoptotic bodies.

The ApopTag® system differs significantly from previously described in situ labeling techniques for apoptosis, in which avidin binding to cellular biotin can be a source of error. The digoxigenin/anti-digoxigenin system has been found to be equally sensitive to avidin/biotin systems. The sole natural source of digoxigenin is the digitalis plant. Immunochemically-similar ligands for binding of the anti-digoxigenin antibody are generally insignificant in animal tissues, ensuring low background staining. Affinity purified sheep polyclonal antibody is the specific anti-digoxigenin reagent used in ApopTag® Kits. This antibody exhibits <1% cross-reactivity with the major vertebrate steroids. In addition, the Fc portion of this antibody has been removed by proteolytic digestion to eliminate any non-specific adsorption to cellular Fc receptors.

Solutions and Reagents:

  • Equilibration Buffer: 3.0 mL -15°C to -25°C
  • Reaction Buffer 2.0 mL -15°C to -25°C
  • TdT Enzyme 0.64 mL -15°C to -25°C
  • Stop/Wash Buffer 20 mL -15°C to -25°C
  • Anti-Digoxigenin-Peroxidase* 3.0 mL 2°C to 8°C
  • Plastic 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 cm2 each when used according to instructions. Reaction Buffer will be fully consumed before other reagents when kits are used for slide-mounted specimens.


  1. Deparaffinize sections in 2 changes of xylene for 5 minutes each, and hydrate with 2 changes of 100% ethanol for 3 minutes each, and 95% ethanol for 1 minute.

  2. Rinse in distilled water.
  3. Pretreatment: Use proteinase K digestion method. Note: for frozen sections or culture cells grown on slides, incubate with 0.2% Triton X-100 in PBS-Tween for 30 minutes is required.
  4. Rinse sections in 2 changes of PBS-Tween 20, 2 minutes each.
  5. Peroxidase Blocking: incubate sections in 3% H2O2 in PBS for 10 minutes to block endogenous peroxidase activity (for frozen section, use 0.3% H2O2 in methanol)
  6. Rinse in PBS-Tween 20 for 3x2 min.
  7. Pre-incubation: incubate sections in Equilibration Buffer for 5-10 minutes.
  8. TdT Reaction: incubate sections in Working Strength TdT Enzyme (Mixture of 100 ul Reaction Buffer and 30 ul TdT Enzyme) for 1 hours at 37°C in humidified chamber.
  9. Stop Reaction: rinse sections in Working Strength Stop/Wash Buffer for 10 minutes.
  10. Rinse in PBS-Tween 20 for 3x2min.
  11. Detection: incubate sections with Anti-Digoxigenin Conjugate (Peroxidase) for 30 minutes at room temperature.
  12. Rinse in PBS-Tween 20 for 3x2min.
  13. Chromagen/Substrate: incubate sections with DAB for 1-2 minutes.
  14. Rinse in tap water.
  15. Counterstain with Gill's hematoxylin for 30 seconds.
  16. Rinse in running tap water for 5 minutes.
  17. Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x3min.
  18. Clear in xylene for 2x5min.
  19. Coverslip with xylene based mounting medium.



Staining Pattern: Nuclear (Search Images)



  1. Gavrieli Y, et al (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119:493-501.

  2. Charriaut-Marlangue C and Ben-Ari Y (1995) A cautionary note on the use of TUNEL stain to determine apoptosis. NeuroReport 7:61-64.

  3. Ay I, et al (2001) Intravenous basic fibroblast growth factor (bFGF) decreases DNA fragmentation and prevents downregulation of Bcl-2 expression in the ischemic brain following middle cerebral artery occlusion in rats. Molecular Brain Res. 87:71-80.