Hello everyone,

I am a researcher at the University of Heidelbeg, Germany. I injected the AAV containing Hes1 shRNA into the adult mice brains and perfused them with PFA after 14days. These brain tissue slices were then stored in Na-Azide (in PBS).

When I stained these tissue slices with DAPI, GFP and Ki67, I always found some problems with Ki67 staining. Initially, I could find only very less Ki67+ cells. Then I increased the permeabilization time (NP40 0.5%) from 10mins to 30 mins.  Even after this, I couldn't find many Ki67+ cells and also the signal is very weak. I don't know where it's going really wrong.

Some of the key steps involved in the immunostaining:

Day 1: 

30 mins- Permeabilization with NP40 0.5% (in PBS)

30mins- Quenching with 0.1M glycine (in PBS)

30mins- Quenching with 50mM NH4Cl (in PBS)

90mins- blocking with 5% FCS (in PBS)

Overnight incubation with primary antibody (In FCS)- mouse GFP(1/1500), rabbit Ki67 (1/100)

Day 2: 

30mins(more than 3times)- washing with PBS

90mins- incubation with secondary antibody

30mins (3times)- washing with PBS

10mins- DAPI staining (1/500)

10mins- washing with PBS

Embed in Mowiol.

Can someone see where I am getting it wrong?

Any kind of suggestion would be greatly appreciated.

Thanks very much.