Hi,
I want to preincubate 20m thick brain cryosections (brain was perfused with formalin) with proteinaseK to stain ssDNA. The problem is that the sections go off the slides by using proteinaseK, I tried:

20 g/ml 20 min, 15 min, 10 min @RT
15 g/ml 15 min @RT
10 g/ml 20 min @RT
50 g/ml 5 min @37°C
...
in 0.1 M PBS

Plus: staining against ssDNA did not really give good results.

Have you got an idea whether section are too thick for proteinaseK incubation? Or maybe for the thickness, one can only use 5g/ml or so?

What about Trypsin - I tried 0,1% but in vain, no specific staining.
Could I use PronaseE?
Maybe I should change PBS?


Thank you!

It looks like you have a too high concentration of Proteinase K. I would try 5g/ml at 37C and try different time points 5 - 30 min.

Also keep in mind that when you get a new batch, you have to test is to get the right conditions ( can differ a bit :( ).

Also let the sections dry well onto the slide before starting the process. If not dried well onto them they might fall off even without proteinase K treatment.

I don't know what slides you are using, but Superfrost Plus from Fisher has worked well for me.

Thx, but I use superfrost plus slides and let them dry before... I try lower concentrations and a HCl preincubation instead. Let's see whether it will work!

I don't know if you have tried an antibody against ssDNA. F7-26 is specific to single-stranded DNA and can be used on both frozen and paraffin sections. Bender MedSystems sells it.

www.bendermedsystems.com/bm_products/MAN/156.pdf