[Histonet] trouble in BrdU IHC for brain samples

[Amos Brooks]

Hi,
     Firstly, it is a really good idea to include a highly proliferative
organ as a control to make sure that the BrdU was successfully injected in
the first place. Intestine usually works great for this. If looking at brain
only there may actually not be any positive staining because there doesn't
happen to be any proliferation going on at the time.
     I think you are on the right track ditching the microwave. There is too
much variability and lack of control. I always use 1N HCl, but 2N might
work. I use Trypsin for retrieval as it has given me the best results so
far. I have heard of others having success with citrate HIER (heat induced
epitope retrieval) but it wasn't as good in my experience. Beyond that, I
would play with the dilution to make sure you have that nailed down. A good
control is critical though. Please drop me a message if you need any more
help. I know it can be frustrating sometimes,

Best of luck,
Amos


Message: 3
Date: Fri, 28 May 2010 11:10:19 +0900
From: "shymaa shawadfy" <sshawdfy <@t> med.kobe-u.ac.jp>
Subject: [Histonet] trouble in BrdU IHC for brain samples
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <20100528021040.33D7724AF36 <@t> smtpgate.kobe-u.ac.jp>
Content-Type: text/plain;       charset="iso-8859-7"

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 ěm 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