Re: [Histonet] Reply double immunostaining on same species, mouse


That's cool and please kindly let me know the detail.

Yes we used PFA to fix tissue and we have both microtome / cryostat to make sections.
the cryostat sections are embeded with O.C.T.
We have several mouse monoclonal antibodies, and I will use only Alexa fluroscence conjugated 2nd antibody rather DAB staining.

Thanks a lot.



发件人: Gayle Callis 
发送时间: 2008-07-14  07:05:57 
收件人: Histonet 
主题: [Histonet] Reply double immunostaining on same species, mouse 
We do double and triple immunfluorescence staining on mouse tissues with all antibodies raised in same host species only on mouse tissues,  rat antiMouse.   It takes some careful blocking and organization, but it works beautifully.  Please contact me and I will discuss the details on how to set up the staining, but supply some details on what you want to see as green or red fluorescence.  This is only done on frozen sections and NOT on formalin fixed paraffin embedded tissue to eliminate autofluorescence. There are several ways to set this up, and we do use some biotinylated primary antibodies along with Molecular Probes Streptavidin Alexa fluor dyes.  Gayle M. CallisHTL/HT/MT(ASCP)Bozeman MT You can still have issues with the second red detection trying to stick to the first AB unless you do some very aggressive blocking.
-----Original Message-----
Sent: Saturday, July 12, 2008 9:58 AM
To: histonet
Subject: [Histonet] Double immunostaining with two same source 1st Antibody
Hi all
Just wonder any one of you tried to perform IHC using two primary antibodies from same source, mice, for example?
The process is that 
Mice anti-A (1st IHC)->secondary antibody labeled with GREEN
-> Mice anti-B (2nd IHC)-> Red fluroscence
then there's no cross reaction.
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