Response to Kate Sunn's colocalization question.

David R. Cool david.cool at WRIGHT.EDU
Tue Oct 26 09:16:33 EST 1999


Kate,

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.

Good luck

David Cool

David R. Cool, Ph.D.
Assistant Professor
Wright State University
Department of Pharmacology & Toxicology
237 Health Sciences
3640 Colonel Glenn Hwy
Dayton, OH 45435-0001

Tel: 937-775-2457
Fax: 937-775-7221

http://www.med.wright.edu/som/academic/pharm/pharm.html
http://hs21311c.pharm.wright.edu/CoolLab/coollab.html





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