[Histonet] GFP antibodies

[Jimmy Lao]

Kristen,
I think you are very close to success since you could
see week signal in the Immunofluorescet staining. As
my experience, you have to strickly keep all afterward
process in Dark after the step you put fluorochrome
conjugated Ab.
My experiment is to use 1st Ab rabbit anti-GFP IgG
fraction from Molecular Probes, the 2ndary Ab is
Oregon Green goat anti-rabbit IgG(H+L). It works.
even double staining with BrdU.
Good Luck!
Jimmy

--- "Rothammer, Kristen"
<Kristen.Rothammer <@t> stjude.org> wrote:
> Hello Everyone-
> 
>  
> 
> I am desperately, and rather unsuccessfully, trying
> to find a better way
> to look at eGFP in frozen kidney sections from mice.
> 
> I have read a wealth of info on histonet regarding
> use of GFP
> antibodies, and I have tried a number of methods,
> but I am still not
> having any luck.
> 
> I have tried the following:
> 
>  
> 
> (1)     looking directly at eGFP in frozen sections
> (sections are just a
> few weeks old) right after 15 min fixation with 4%
> paraformaldehyde
> (fresh 16% paraformaldehyde diluted in PBS),
> followed by washes in
> PBS--> no GFP is observed
> 
> (2)     looking directly at eGFP in same frozen
> sections without
> fixation, coverslipping in PBS --> no GFP is
> observed
> 
> (3)     fixation in paraformaldehyde as described
> above followed by a
> GFP antibody conjugated to a fluorochrome -->  very
> little signal is
> observed
> 
> (4)     same fixation, followed by incubation with
> GFP antibody, follwed
> by biotinylated anti-rabbit, and finally by
> streptavidin-Alexa 568
> 
> *         This method resulted in EXTREMELY high
> background.  I tried to
> block endogenous biotin by using both the egg/milk
> protocol and
> biotin/avidin drops from Vector Labs, both with
> little success.
> 
>  
> 
> I mount my slides with Flouromount, do NOT seal with
> nail polish, and I
> do not do antigen retrieval (this is not necessary
> for frozen sections,
> right?).
> 
>  
> 
>  
> 
> Any input would be greatly appreciated!
> 
>  
> 
> Thank you!
> 
>  
> 
> Kristen Rothammer
> 
> St. Jude Children's Research Hospital
> 
> Department of Biochemistry
> 
> Kristen.rothammer <@t> stjude.org
> 
>  
> 
> 


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