We are using TUNEL kit from promega in free floating brain sections previuosly fixed in 4% PFA. We had used either PBS + Tween 20 or proteinkinase K as permeabilizer and we had followed the manufacturer instructions and we can not see any signal. Can someone please give me a piece of advice?
Thank you for your answer. Actually you are right, the promega tunel kit is intented for use with cells in suspension as well as for paraffin-embeded tissues whose thickness is about 10 um, ours sections are 50 um thick and we adjusted the amount of reagents according to the manufacturer instructions regarding surface area and we have no signal at all. We think that maybe there is a need for more time in any of the steps. Anyway, we do appreciate your thoughts.
Hi Alejandro,
I just saw your post about TUNEL.... I bought the same TUNEL detection kit from Promega and try to detect apoptosis in the brain. Unfortunetely in both cases - for mounted and free-floating sections I did not see apoptotic nuclei (except boosted with DAPI signal, so I guess it was I used too much concentration). I wonder if you overcome the difficulties?? Please let me know. I use rat and mice brain tissue 4% PA fixed, sections are 40 um thick. As positive control I used mouse brain after tMCAO and also tissue digested with DNaseI as they suggest in the protocol.
Dear Alejandro and Grazyna,
Did you solve that problem? I use free-floting sections 40 um thick. In my protocol there are almost all steps in +37, and sections at the end are destroyed a lot...will try to put sections on the glass and stain by puppeting, may be this process will be better. If you found some decision how to do this staining better, please, share. Kind regards, Polina
Hi everyone,
I had the same problem and was solved by this article. I tried their method, it works beautifully. http://www.ncbi.nlm.nih.gov/pubmed/11532339
I have also attached my optimized protocol here that will save you a lot of reagent. This should give you very reliable and strong TUNEL signal. I only tried 30 um sections but I am guess it should also work for 40 um sample.
Good luck! -Lihong
Dear Lihong,
I will try your method, hope I will have a good result with that. Thank you for sharing!
Kind regards,
Polina
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