[Histonet] Re: Is TUNEL a waste of time? 3% citric acid step

[Walters, Katherine S]

Hi Sarka,

I pulled up your article and I noticed that you used a 3% citric acid
step before the TUNEL stain for minimizing non-specific staining.  What
is the purpose of that step?  (I tried to pull up the article that you
referenced there, but our account only goes back to 1998).

Thanks for any information you can give me.

Kathy

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Sarka
Lhotak
Sent: Tuesday, April 12, 2005 4:11 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Re: Is TUNEL a waste of time?

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
caspase-3.


Sarka Lhotak
McMaster University, Hamilton, Ontario
  



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