[Histonet] re: BrdU on cultured cells

[Kevin Conway]

Hi Steve,

In addition to permeabilizing (we used .15% Triton X-100, no reason the saponin wouldn't also work), I added a denaturation step (x 10 min in ?2 N HCl (aq)- I will double-check the [HCl] and let you know for sure if you don't have a protocol. Just helps to keep the histones away :)

Also, I'm not sure what antibody you're using, but we always had great success with the G3G4 Mab from DSHB.
Good luck,

Kevin
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Date: Wed, 7 Dec 2005 12:31:10 -0800 (PST)
From: Steven Coakley <sjchtascp <@t> yahoo.com>
Subject: [Histonet] Brdu staining fixed cultured cells.


Good afternoon everyone,
   
  I'm working witha scientist who is attempting to stain previously fixed cultured cells in "wells" and cover slips with anti-Brdu.  He has fixed the cells 1st in methanol, air dryed, then in 10%formalin/PBS and again air dryed.  Our anti-BrdU/DNAase works great on tissue but not on the fixed/air dryed cultured cells.  We tried a 30 minute 0.1% Saponin step to help permeablize the cells but w/o success.  Any ideas.
   
  Steve

			
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Kevin Conway, PhD
Assistant Professor
Department of Anatomy
Canadian Memorial Chiropractic College
416-482-2340 x.258
kconway <@t> cmcc.ca