Hi TF,
we do IF with two 1.ABs out of mice using a sequential protocol from
abcam: www.abcam.com/technical
We perform a second blocking step with serum (higher conc. than the
first one) after the first Fluorescence was added, do not perform
another retrieval procedure (my ABs need the same) and perform the
following incubation steps in the "dark" (we switch the light off and
cover the slides after adding the ABs) to prevent fading.
Until now, I was sure, this procedure works but maybe the two antigens
don't colocalize but it is just a staining artifact ;-)
Hope this helps,
Frauke
Zitat von TF <ti...@foxmail.com>:
Thanks all, Merced M Leiker is right: the double IHC procedure using
two primary antibodies from same species.
Some expert just sent me their procedure of Heat-interval based
inactivation of the primary antibody for first antigen.
I am just seeking for a chemical way at room temperature.
2009-05-18
TF
发件人： Rene J Buesa
发送时间： 2009-05-18 22:31:30
收件人： tifei; gu.lang; Merced M Leiker
抄送： histonet
主题： Re: AW: [Histonet] Chemicals that inactivate the primary antibody
I am completely confused.
IF you have an epitope in the tissue and you react with it an
specific antibody "that is it". The epitope will have reacted and I
do not see any way in which you can add another antibody to that
initial epitope. From the reactive point of view, the epitope is
"blocked" to react with another, unless the reaction is not totally
specific and "there is room for another".
I just cannot imagine a way of doing this.
René J.
--- On Mon, 5/18/09, Merced M Leiker <lei...@buffalo.edu> wrote:
From: Merced M Leiker <lei...@buffalo.edu>
Subject: Re: AW: [Histonet] Chemicals that inactivate the primary antibody
To: ti...@foxmail.com, gu.l...@gmx.at
Cc: histonet@lists.utsouthwestern.edu
Date: Monday, May 18, 2009, 9:42 AM
Hi TF and all interested,
I think I know what you want, but unfortunately I don't know how to answer
your question (it is something I'd like answered myself!!) To re-word for
the sake of all interested:
You want to perform double-immunofluorescent staining using 2 primaries
that were raised in the same species.
An additional question for clarification is: Do you want to do this on
paraffin or frozen sections?
Maybe someone can help figure it out...
Merced
--On Sunday, May 17, 2009 2:16 AM +0800 TF <ti...@foxmail.com> wrote:
But the heat also damage the fluorescnece...
u need specific amplication kit anyway.
2009-05-17
TF
发件人： Gudrun Lang
发送时间： 2009-05-17 02:00:18
收件人： ti...@foxmail.com
抄送： histonet@lists.utsouthwestern.edu
主题： AW: [Histonet] Chemicals that inactivate the primary antibody
For doublestaining the primary undergoes denaturation through a second
HIER-step. The second secondary ab doesn't bind to the first primary.
Therefore the binding sites must have been destroyed by heat in
retrieval-buffer.
Gudrun
-----Urspr黱gliche Nachricht-----
Von: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von TF
Gesendet: Samstag, 16. Mai 2009 18:48
An: Histonet@lists.utsouthwestern.edu
Betreff: [Histonet] Chemicals that inactivate the primary antibody
Hi all, just wonder what kind of treatments/chemicals can complete block
the binding of 2nd antibody to binded primary antibody on antigen?
I tried HCl , but does not damage all the primaryantibody binding sites -
i can still see staining pattern finally.
2009-05-17
TF
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Merced M Leiker
Cardiovascular Medicine
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State University of New York at Buffalo
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