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Dear Patty,

I routinely use 293HEK cells. They have a bit of autofluorescence (mainly
green) fixing either with 4% paraformaldehyde or cold acetone. For each
experiment I always set up a control slide in which I omit the primary and
secondary antibodies. I consider such a signal background due to
autofluorescence of the cells. I get a rid of the autofluorescence signal
by using the black level control. Then, I will use these settings to
acquire all the images of that experiment.

I hope this helps.
Francesca

At 14:31 29/04/03 -0400, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Has anyone had experience getting rid of autofluorescence from 293
>cells?  Two of my colleagues are trying to perform immunofluorescence on
>293 cells transfected with WISP3 and LRP5 respectively.  However, the
>control cells and transfected cells have high levels of
>autofluorescence.  Has anyone else experienced this with this cells
>line?  Any suggestions on how to get rid of the background staining?
>Both colleagues see this with paraformaledehyde and methanol fixation
>protocols, and the cells have been blocked with BSA and/or serum.
>Thanks,
>
>Patty Conrad
>621 BRB/Dept. of Genetics
>CWRU School of Medicine
>10900 Euclid Ave
>Cleveland, Ohio 44106-4955
>
>Phone:  (216) 368-0305
>FAX:    (216) 368-3432

--------------------------------------------------------------

Francesca Magnani
Biochemistry Department
Trinity College
Dublin 2
Ireland

phone: 00353 1 6082445
fax: 00353 1 6772400
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