I was wondering if anyone has had any experience with bleed through of
fluorescence from the EGFP channel, when looking at an antibody on the red
channel? I have transfected cells with the EGFP fusion constructs and then
wish to look at colocalision with another protein by immunocytochemistry. I
am using an ALEXA-594 secondary antibody, but I cant seem to detect an
intense specific red signal as the green fluorecence is bleeding through to
the red on the confocal. Has anyone had this happen to them, how did you
overcome this problem?
I am trying to fix (in 2%PFA) at 4 degrees, and also was with 0.1% sodium
borohydride to see if this helps, but dont know the results as yet.
If anyone could help I would greatly appreciate it.
Kathryn Sunn BSc (Hons)
Bone and Mineral Research Program
Garvan Institute of Medical Research
St Vincent's Hospital Tel: (02) 295 8260
Sydney NSW 2010 Fax: (02) 295 8241
Australia email: k.sunn at garvan.unsw.edu.au