AW: Re: AW: [Histonet] Chemicals that inactivate the primaryantibody

[Gudrun Lang]

Hi Frauke,
I've read the abcam protocol. They don't indicate what species the ab's =
are
from. Or I've overlooked it.
What is the reason, why the second secondary ab doesn't bind to the =
first
primary ab?=20

Gudrun

-----Urspr=A8=B9ngliche Nachricht-----
Von: Dr. Frauke Neff [mailto:nefff <@t> staff.uni-marburg.de]=20
Gesendet: Montag, 18. Mai 2009 18:17
An: tifei <@t> foxmail.com
Cc: Rene J Buesa; gu.lang; Merced MLeiker; histonet
Betreff: Re: Re: AW: [Histonet] Chemicals that inactivate the
primaryantibody

Hi TF,
we do IF with two 1.ABs out of mice using a sequential protocol from =20
abcam: www.abcam.com/technical
We perform a second blocking step with serum (higher conc. than the =20
first one) after the first Fluorescence was added, do not perform =20
another retrieval procedure (my ABs need the same) and perform the =20
following incubation steps in the "dark" (we switch the light off and =20
cover the slides after adding the ABs) to prevent fading.

Until now, I was sure, this procedure works but maybe the two antigens =20
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 =20
>  two primary antibodies from same species.
>
> Some expert just sent me their procedure of Heat-interval  based  =20
> inactivation of the primary antibody for first antigen.
> I am just seeking for a chemical way at room temperature.
>
>
> 2009-05-18
>
>
>
> TF
>
>
>
> =B7=A2=BC=FE=C8=CB=A3=BA Rene J Buesa
> =B7=A2=CB=CD=CA=B1=BC=E4=A3=BA 2009-05-18  22:31:30
> =CA=D5=BC=FE=C8=CB=A3=BA tifei; gu.lang; Merced M Leiker
> =B3=AD=CB=CD=A3=BA histonet
> =D6=F7=CC=E2=A3=BA 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  =20
> specific antibody "that is it". The epitope will have reacted and I  =20
> do not see any way in which you can add another antibody to that  =20
> initial epitope. From the reactive point of view, the epitope is  =20
> "blocked" to react with another, unless the reaction is not totally  =20
> specific and "there is room for another".
> I just cannot imagine a way of doing this.
> Ren=A8=A6 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
>>
>>
>>
>> =B7=A2=BC=FE=C8=CB=A3=BA Gudrun Lang
>> =B7=A2=CB=CD=CA=B1=BC=E4=A3=BA 2009-05-17  02:00:18
>> =CA=D5=BC=FE=C8=CB=A3=BA tifei <@t> foxmail.com
>> =B3=AD=CB=CD=A3=BA histonet <@t> lists.utsouthwestern.edu
>> =D6=F7=CC=E2=A3=BA 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=FCngliche 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
>
> No trees were harmed in the sending of this email.
> However, many electrons were severely inconvenienced.
>
>
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