[Histonet] GFP antibodies

[Rothammer, Kristen]

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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