Re: Re: AW: [Histonet] Chemicals that inactivate the primary antibody

[TF]

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 <leiker <@t> buffalo.edu> wrote:


From: Merced M Leiker <leiker <@t> buffalo.edu>
Subject: Re: AW: [Histonet] Chemicals that inactivate the primary antibody
To: tifei <@t> foxmail.com, gu.lang <@t> gmx.at
Cc: histonet <@t> 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 <tifei <@t> 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： tifei <@t> foxmail.com
> 抄送： histonet <@t> 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-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von TF
> Gesendet: Samstag, 16. Mai 2009 18:48
> An: Histonet <@t> 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
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet



Merced M Leiker
Cardiovascular Medicine
348 Biomedical Research Building
State University of New York at Buffalo
3435 Main St, Buffalo, NY  14214
leiker <@t> buffalo.edu
716-829-6118

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However, many electrons were severely inconvenienced.


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