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GFP bleed thru in double labelling

Beat Ludin bludin at lis.ch
Thu May 18 08:33:02 EST 2000

What filters/settings are you using? Do you record the two channels 
simultaneously (emission filtering only) or sequentially (excitation 
and emission filtering)?


>Dear All
>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

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