GFP bleed thru in double labelling

Jos Broers Jos.Broers at molcelb.unimaas.nl
Wed May 24 10:42:21 EST 2000


Dear Kathryn,

If you use the  right filter set bleed through of EGF into the red channel
should not be a serious problem. We use a rather narrow bandpass filter for GFP
(the same one which we use also for FITC) and use TexasRed with a LP filter for
the immunolabeling and encounter no serious bleed through unless the GFP signal
is extremely bright.
Fixation with PFA can cause autofluorescence, but you easily distinguish this
from the GFP signal , since you will notice that also in areas of the cell where
you don't have GFP signal.
Kind regards

Jos
Kathryn Sunn wrote:

> 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.
>
> Thanks
> Kate
>
> ___________________________________________________________________
> 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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