RE: [Histonet] TUNEL staining
I used Roche In situ cell death detection kit Cat No 11 684 817 910. I modified protocol by permeabilizing paraformaldehyde-fixed frozen sections with Triton 0.5% for 10 min at RT, and reducing TdT concentration 1:10 from manufacturers recommendation. With that I was able to obtain 100% positive cells in DNase I treated samples, thus ensuring efficient permeabilization and activity of enzime.
I might still be artificially detecting an excessive number of cells with these conditions, although numbers I found in mouse kidney are in agreement with what is described in literature (an indeed very low number of positive cells). As a way to increase especificity I always counterstain with DAPI, and most green TUNEL+ cells have a more whitish nucleus when seen with DAPI filter. Still you can have cells with DNA damage unrelated to apoptosis and they will be TUNEL+. Because of all this issues I wanted to confirm results with an additional technique such as Active Caspase-3, but before assuming unspecific TUNEL staining in all Casp3 negative but TUNEL+ cells, I wanted to make sure I got optimal working conditions for Anti-active Caspase-3 Ab.
After optimizing fixation and permeabilization conditions to get good positive controls with DNase treated samples you can always titrate TdT concentration to obtain maximum signal to noise ratio as shown in the paper I mentioned before.
If you need any further details of have any suggestions please tell me.
"Kalleberg, Kristopher" escribió:
I am currently performing TUNEL on human skin post UV irradiation and
have had inconclusive results. I was using the Promega TUNEL assay kit
and also tried a formamide induced TUNEL assay with differing results.
I also have read in the literature that the kit and most TUNEL assays
are nonspecific (necrotic cells also show positive staining). The
formamide induced protocol that I have only tried twice has shown no
positive staining yet. I was just wondering which protocol you use for
TUNEL and how certain are you that it is specific for apoptotic cells?
Maybe we can help each other.
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