I was wondering if there may be a problem of permeabilisation of cells. I am working on mice hippocampus (pups) and I need to make a staining for a phoshoprilated nuclear protein. Since the antibody showed to work before, I was wondering if somebody had the same problem and how did you fix it.
Thanks
Hi Stefano;
Maybe you can give us more details about this, because there are so many things that can go wrong is just impossible to know with the info you´re giving here.
how the specimens were obtained, fixation... and so on.
hope someone can help you!
Hi Oscar, thank you for your answer. Sorry You are right, I focused so much on the problem that I ignored to tell the procedure.
I prepared organotipic slices for hippocampus and I had them attached to a Millipore membrane. At the moment of the staining I treated them with PFA (4%) and I washed them with PBS three times for 10 minutes previous permeabilisation with TX 100 (0.4%) for 90 minutes. After that I incubate 1 hour in blocking solution (10%) NGS in PBS (NH4Cl 50 nM was in the blocking solution) and I incubated 72 hours with the primary antibodies (Cell Signaling, Sigma and Millipore).
Secondary antibody was left in incubation ON (1:500): I used a goat-antirabbit and anti-mouse 2ab from (invitrogen). I washed 3x then for 1 hour.
I Stefano,
Hopefully you already fix this issue but otherwise what was the problem? You couldn't see signal? Was it not specific or background signal was to strong?
Cheers,