apoptosis TUNEL assays in mice Kidney - False positive (Dec/08/2005 )
I'm a PhD student trying to optimise TUNEL assays on mice kidney sections. I'm getting a very high false positive (80%) in wild type using the normal TUNEL protocol with proteinase K pretreatment. The sections are formalin fixed and mounted in paraffin. I have already reduced TdT to 20% from the recoended 30% on the Chemicon apoptag peroxidase kit.
Have any of you come across this issue? Any suggestions around this problem?
I don't know anything about mice kidney sections, but I have tried to do a TUNEL test on porcine corneas and I also got a very high false positive ratio. In my case it was due to folding of the cornea. Cells died due to mechanical stress.
How do you treat the kidneys before you fix them??? Anything that could stress the cells???
In your case, since you use a commercial kit, you can also try to call technical service from Chemicon, they are usually very, very helpful.
its fixing them in formalin and they are sectioned in paraffin. this is most recommended in using TUNEL assays so that bit should be fine, it could be pretreatment in proteinase k that leads to false positive. I might try pretratment of slides with DEPC to get rid of endogenous nucleases tomorrow. hoping that it works. If not then I will mail chemicon. Thanx for the advise.
We are also getting high positive staining in a Chemicon TUNEL assay using mouse kidney and heart sections. Our samples are frozen sections. We have tried diluting the TdT enzyme to 1:100 (the recommended dilution is about 1:3) and reducing the incubation time, but this has not helped. Sony, have you found a solution to this problem yet?