Re: Primary antibody incubation
As far as I know, overnight 4C is about equal to 2 hours RT. At least
spoken in terms of energy. As we are generally applying a 60 min incubation
for our primaries, overnight 4C usually yields a stronger staining.
Furthermore, overnight 4C suppose to be better for low-affinity primaries,
as they have longer time to bind properly to the antigen. I don't have any
proof on this.
I agree with Vikky that their is no universal "best".
To complete the story on primary incubation: to my opinion if you mention a
dilution for a certain antibody, you should also mention the
time/temperature and detection system that was used.
Chris van der Loos
Dept. of Cardiovascular Pathology
Academic Medical Center
Amsterdam - The Netherlands
Fernando and Vicky wrote:
>Date: 28 Mar 2003 09:33:18 -0600
>From: Victoria Baker
>Subject: Re: Primary antibody incubation
>My answer to this is it depends, I don't believe there
>really is a universal "best". I have worked in
>clinical, industrial and currently in academia, I
>handle human and animal tissue/cells and do a large
>variety of IHC/IF/ISH/FISH work. I will tell you now
>that primary incubation times are never written in
>stone because they are only ONE part of the procedure.
> Each lab has to work out its own conditions based on
>the variables they have to work with. Both the
>conditions and variables differ from lab to lab.
>In my lab currently we run PCNA, bcl-2, Cyclin D1,
>GSTpi, 3-clones of p53, and BrdU at one to two hours
>incubation at RT.
>All NFkB subunits, 8oxodG, BPDE, Cox 1 & 2, p21,
>3-nitrotyrosine and MGMT overnight at 4 C.
>Just name a few.
>Institute for Cancer Prevention
>Valhalla, New York 10595
- --- Fernando Capela e Silva wrote:
> What is the best in immnuohistochemestry:
> - primary antibody incubation overnight at 4 degrees
> - primary antibody incubation 30-60 min at room
> temperature ?
> With compliments
> * * * * * * * * * * * * * * * * * * * * * * * * *
> Fernando Capela e Silva
> Laborat#243#rio de Biologia da Conserva#231##227#o
> Departamento de Biologia
> Universidade de ...vora
> Apartado 94
> 7002-554 ...vora
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