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Hi
I have recently embarked on flow cytometry and am interested in 
combining that with looking at intracellular markers by microscopy. 
Does anyone know why the commonly used flow dye PE (phycoerythrin)  is 
rarely (to never)  used for immunofluorescence microscopy? I went ahead 
and stained with a PE labeled antibody and found a confocal that should 
have allowed me to combine the right ex/em wavelengths to see it and 
could not (although it was easily seen by flow cytometry). The 
excitation spectra of PE is very broad and that would make it hard to 
combine with other dyes but is there something else that makes it 
unsuitable for immunofluorescence. A flow person suggested that flow 
cytometry is so much more sensitive that you might not expect to see 
it. Perhaps someone has direct experience and can advise me.

Thanks

Dorothy

PS   When I stain cell surface labeled cells using my normal 
immunofluorescence protocol for an intracellular antigen developed for 
microscopy  I do seem to get a very high background in the flow 
cytometer with my normal rabbit serum control. By microscopy the 
difference is like night and day but  by flow there was a huge 
background so any insight into why or how to reduce that would be nice 
too (this was using  an anti-rabbit Alexa 488 secondary) Merci!
____________________________________
Dorothy I. Mundy, Ph.D.
Assistant Professor
Department of Hematology Oncology
5323 Harry Hines Blvd.
Dallas, TX 75390-8565

Phone: 214-648-1909
[log in to unmask]


***********************************************
Dorothy I. Mundy, Ph.D.
Assistant Professor
Department of Hematology Oncology, NB7.116
University of Texas Southwestern Medical School
5323 Harry Hines Blvd.
Dallas, TX 75390-8565

Phone: 214-648-1909
[log in to unmask]