[Histonet] pErk/Ki67 doublelabeling

[Neil M. Fournier]

I am working with fixed (10% buffered formalin) rat brain sections and I have
had some difficulty getting pErk/Ki67 immunofluorescent colabeling to work. I
get beautiful Ki67 labeling but absolutely no pErk labeling anywhere. I am
using mouse monoclonal pErk (Cell Signaling) and rabbit monoclonal Ki67(Vector)
antibodies. Primaries are incubated simultaneously for 48 hr at 4 degree C. I am
using Alexa 488 and 546 secondary antibodies, which are always given
simultaneously during staining for 3 hr at RT.

Monolabeling with pErk is fine with other brain sections (the only difference is
that they were sectioned on a freezing microtime versus a vibratome in the
present study). However, the only labeling I see with pErk in my sections is
nonspecific labeling of capillary beds throughout the section. I have tried
staining both with and without citrate buffer antigen retrieval and there is no
difference.

Does anyone have any suggestions or recommendations?

Neil