[Histonet] TUNEL

[Sarka Lhotak]

Hi Melissa,

I use the Trevigen system with great success on mouse tissue using
biotinylated dNTPs, Streptavidin HRP and NovaREd. It should work just
as well with fluorescence. Mouse thymus is a great positive control.
My protocol is as follows:

-Xylenes, ethanols, water.

-Proteinase K 30 minutes at 37C

-Endog. peroxidase quenching: 45 ml methanol+5ml 30% H2O2, 5 min.

-1x labeling buffer, 5 min

-Reaction mixture: 1x labeling b. 50ul + TdTdNTP mix 1ul+ 50xCo2+ 1ul +
TdT enzyme 1ul. 1hr at 37C.
 
-1x Stop buffer, 5 min RT
Then I use the Streptavidin HRP and Nova Red that I use for IHC,
exactly the same way.

I found that the Endog. peroxidase quenching step was ESSENTIAL for
this reaction to work, i.e. probably doing more than just quenching,
without that there was NO reaction at all.  

Hope this helps somewhat, don't hesitate to contact me if you have more
questions. The beauty of the Trevigen system is that you can buy
individual reagents, no need to be paying big bucks for a full kit.
(No commercial interest in Trevigen :), just a happy customer).

Sarka Lhotak

McMaster University, Hamilton