[Histonet] GFP antibodies

From:"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 PBSà no GFP is observed

(2)     looking directly at eGFP in same frozen sections without fixation, coverslipping in PBS à no GFP is observed

(3)     fixation in paraformaldehyde as described above followed by a GFP antibody conjugated to a fluorochrome à  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



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