Re: RE: [Histonet] Double immunostaining with two same source 1st Antibody


Dear Anatoli:

Thanks very much for your suggestions.

Yes I am using Abcam rat monoclonal Brdu antibody, and I also have tried mouse monoclonal.

SOme people in my lab suggested that without HCl, you may get better DAPI co-staining after BrdU staining, however sometimes you can not get BrdU staining...
We dont know why. Citrate buffer might be able to break some DNA helix during heating process (30 min with 95 degree). But I was just following the protocol.

Next time I will try it. - -




发件人: Anatoli Gleiberman 
发送时间: 2008-07-14  21:57:39 
主题: RE: [Histonet] Double immunostaining with two same source 1st Antibody 
For BrdU staining boiling in citrate buffer is good enough to denature DNA - you don't need to treat sections  with HCl after that. I did it on both paraffin and cryo sections with the same good results. Another suggestion - for BrdU staining use rat monoclonal against BrdU from Apcam - they are much more stronger and cleaner than any mouse monoclonal I have used so far.
Anatoli Gleiberman, PhD
Director of Histopathology
Cleveland Biolabs, Inc
73 High Street
Buffalo, NY 14203
phone:716-849-6810 ext.354
e-mail: AGleiberman@cbiolabs
-----Original Message-----
From: [] On Behalf Of tf
Sent: Sunday, July 13, 2008 10:02 PM
To: Neil Fournier; histonet
Subject: Re: [Histonet] Double immunostaining with two same source 1st Antibody
Some one suggested a "citrate buffer" treatment before 2nd IHC.
However he als suggested be careful about losing anti-gen in this step.
Actually I used this step for BrdU staining pretreatment before adding HCl to broken DNA helix (brdu can be taken by cell and synthsized into DNA).
It seems that citrate buffer has more function than simply the antigen retrieval.
Can anyone comment on this further?
发件人: Neil Fournier 
发送时间: 2008-07-14  03:26:20 
主题: [Histonet] Double immunostaining with two same source 1st Antibody 

I am extremely interested in the suggestions you receive. I would like to do a similar procedure. If possible, would you be able forward them to me.
Much appreciated, 
Neil M. Fournier
Department of Psychology
University of Saskatchewan
9 Campus Drive
Saskatoon, SK, Canada
S7N 5A5
Phone:  306-966-4024
    Fax:  306-966-6630 

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