AW: [Histonet] Chemicals that inactivate the primary antibody

[Dr. Frauke Neff]

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 <tifei <@t> 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 <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
>> _______________________________________________
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>> Histonet <@t> lists.utsouthwestern.edu
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>
>
>
> 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
>
> No trees were harmed in the sending of this email.
> However, many electrons were severely inconvenienced.
>
>
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