Re: Primary antibody incubation

From:Chris van der Loos

Dear Fernando,
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.
 >Vikki Baker
 >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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