GFP bleed thru in double labelling

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Thu May 18 04:16:43 EST 2000


Kathryn Sunn <k.sunn at garvan.unsw.edu.au> wrote:
> 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 cannot confirm that. I used various (green) GFPs and could successfully
colocalize them with Alexa-594 without any bleedthrough. I fix my cells
in PBS / 5% Sucrose / 3% PFA at room temperature for 20 minutes and mount
them in 60% glycerol in PBS without any antifade agent.

--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP4 */
/* "Science is the game we play with God to find out what His rules are."  */




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