| 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