Response to Kate Sunn's colocalization question.
David R. Cool
david.cool at WRIGHT.EDU
Tue Oct 26 09:16:33 EST 1999
I have been creating fusion proteins with EGFP, EYFP and prohormones
for over a year and colabeling with both rhodamine and texas red.
Like the other response, I fix in 2% PFA with 0.1% triton X100. After
staining, mount using Biomeda's Gel Mount. I have found that it is
very good at preserving fluorescence. I have some slides dating back
6 years that still have their fluorescence.
Two things to be careful about though. 1) fix the cells with a good
paraformaldehyde (I use EMS's EM grade PFA). A friend in another lab
makes his own PFA and the GFP tends to quench over a few weeks time.
and 2) be careful of the UV light on your samples. I have seen some
very fast quenching with the UV filter on our microscope. Even the
Rhodamine filter can diminish the GFP fluorescence.
David R. Cool, Ph.D.
Wright State University
Department of Pharmacology & Toxicology
237 Health Sciences
3640 Colonel Glenn Hwy
Dayton, OH 45435-0001
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