[Histonet] trouble in BrdU IHC for brain samples

[shymaa shawadfy]

Dear Histonet members

 

I am having great trouble in my BrdU IHC in mice brain samples. I am trying
to establish a protocol for paraformaldehyde fixed - paraffin embedded
sections. I cut samples at 6 μm thickness. 

Few times I got good nuclear stain but other times using the same protocol I
fail to get any signal. In addition to high background

I used microwave oven for antigen retrieval (citrate buffer) in addition 2 N
HCl for 60 min at 37 degrees.  Using 2N HCl alone produced weak signal. I
also tried combining pepsin digestion with HCl but I did not get any signal
and my tissues were over digested. And now I think that I will stick to
water bath at 98 degrees for 40 min instead of the microwave. 

Blocking is 2 % BSA + 2% NGS in PBS T 

I used BD bioscience anti BrdU  at 1:100 dilution , then biotin conjugated
anti mouse , vector ABC kit and color development with DAB with Nickel
enhancement.

 

Do you have any suggestions?

 

I read that we can also use 0.07 N NaOH to enhance the antibody reactivity;
can you please tell me the protocol? 

 

 Thank you 

 

Shymaa