[Histonet] Re: Is TUNEL a waste of time?

From:"Sarka Lhotak"

Dear Toshi,
TUNEL is not a waste of time, just don't use the Roche kit! I had the
same problems with it: lots of normal looking nuclei staining positive.

Now I am using the Trevigen TACS kit (in Canada sold through
BIO/CAN)and it works great. You can buy individual components, no need
to get the whole kit each time you run out of a component. I buy the
TdT enzyme, biotinylated dNTP nucleotides, Co 2+ cations, Proteinase K,
TdT Labeling buffer and TdT Stop buffer. I follow their protocol up to
the Stop buffer step, then I vizualize the incorporated biotinylated
nucleotides either with my usual IHC Streptavidin-peroxidase, Nova-Red
incubations, or for fluorescence, with Streptavidin-Alexa from
Molecular Probes.

The staining is very clean with nuclei of apoptotic morphology staining
positive. You can check my double immunofluorescence for TUNEL and
cleaved caspase-3 in mouse thymus in our recent paper: Circulation
2005;111:1814-1821, Supplemental on-line Figure 5. TUNEL specifically
stained nuclei in cells that had cytoplasm positive for cleaved

Sarka Lhotak
McMaster University, Hamilton, Ontario

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