DNA fragmentation

[Veronica]

Hello,

this is not really an ICH question, but I ask anyway.
Earlier I have been using the TUNEL assay to detect DNA fragmentation in paraffin tissue sections, but with weird results. Now, I would like to try in situ nick translation with DNA polymerase (from E coli) instead. Does anyone have a good protocol for this? What is your experiances of this method?

[richard03]

Try Chemicon Apop Tag kit and it works great. Roche kit also good but sometimes you need to worry about endogenous biotin.

http://ihcworld.com/apoptosis_assay.htm

[dutchmaninvienna]

Hi Veronica,

My lab has a long tradition with apoptosis, TUNEL and ISNT (see for instance     
Gold R, Schmied M, Rothe G, Zischler H, Breitschopf H, Wekerle H, Lassmann H.   
Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections. J Histochem Cytochem. 1993 Jul;41(7):1023-30 or my latest publication; Bauer J, Elger CE, Hans VH, Schramm J, Urbach H, Lassmann H, Bien CG. Astrocytes are a specific immunological target in Rasmussen's encephalitis. Ann Neurol. 2007 Jul;62(1):67-80.).

I think that if you have "weird" results with your kit, you probably will have the same problems when doing ISNT. During the last years we have been using the Roche kit and I think it is working excellent. Note however that both ISNT as well as TUNEL cannot discriminate between apoptosis and necrosis. Therefore, if you want to look at apoptosis, I would in addition use an activated caspase-3 staining. If you do both, you may find that they will may not have the same staining pattern, one of them may stain more, the other less because they label apoptotic cells at different stages of the apoptotic pathway. Maybe if you tell us what you mean with weird results we can help you in solving your problems. It may also happen that your TUNEL is positive and your caspase-3 negative. This means that your cells either are necrotic, or they follow a non-caspase mediated apoptotic pathway

jan