Hi,

I don't know whether you avoided the permiabilization or not. As it is in extra-cellular, one should avoid permiabilization or use mild permiabilization. and do blocking properly an dont keep the primary antibody overnight.

Hi,

Prakash and Hussein.

Thank you for your responses.

Even though my tissue is fixed in ethanol, i do  a short antigen retrieval step with pepsin for 5 mts, and incubate with primary ab. overnight after the blocking step.

There are  no extra permeabilizations step except washes in PBST.

i will try incubating in room temp. for 3-4 hrs.

Hopefully that will help.

I have also tried fixation in 4% PFA, but with that the staining in the control mice looks good ,but we dont see intracellular staining in the mutant mice tissues, as expected. in this case antigen rerieval is with pepsin for 30 mts.

With ethanol fixation it is the opposite, where we see intracellular staining in both control and mutant.

So, in both the cases, the control and mutant tissue looks same.

Thanks for yours help