Apoptosis Detection Using ApopTag Peroxidase In
Situ Apoptosis Detection Kit from Chemicon (S7100)
Apoptosis Detection/TUNEL Staining Service
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.
- 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
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.
Rinse in distilled water.
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
Rinse sections in 2 changes of PBS-Tween 20, 2 minutes each.
incubate sections in 3% H2O2 in PBS for 10 minutes to block
endogenous peroxidase activity (for frozen section, use 0.3%
H2O2 in methanol)
in PBS-Tween 20
for 3x2 min.
incubate sections in Equilibration Buffer for 5-10 minutes.
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
rinse sections in Working Strength Stop/Wash Buffer for 10 minutes.
incubate sections with Anti-Digoxigenin Conjugate (Peroxidase) for
30 minutes at
PBS-Tween 20 for 3x2min.
incubate sections with DAB for 1-2 minutes.
in tap water.
with Gill's hematoxylin for 30 seconds.
in running tap water for 5 minutes.
Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x3min.
in xylene for 2x5min.
with xylene based mounting medium.
Staining Pattern: Nuclear
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.
Charriaut-Marlangue C and Ben-Ari Y (1995) A cautionary note on
the use of TUNEL stain to determine apoptosis. NeuroReport
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.