The likely cause is the filters in the confocal. Look at the emission
wavelength of both the EGFP and the ALEXA-594 (remember there are a
whole bunch of different varieties that emmit at different wavelengths
even within the same characterized name so check).
www.omegafiters.com or www.molecularprobes.com (good places to
I have found EGFP excited at from as low as 350 to a max of 525 with a
peak at somewhere just under 500nm.
The EGFP I have found emmits between 480 all the way up to 600nm with
a peak somewhere near 510 nm.
The Alexa 594 can be excited way down below 350 nm all the way up to
600 nm with a peak at 590 ish.
The Alexa 594 emmits somewhere between 575 and over 700 nm with a peak
somewhere near 615 nm.
To get rid of the GFP bleed through make sure the emission filter for
the Alexa 594 is over 600. You loose some signal but you cut down the
bleed through from GFP.
The alternative is to move the excitation of the Alexa up to above
525nm. Therefore you do not excite much EGFP and you still get Alexa
Since you mentioned colocalization I encourage you to check out
www.bitplane.com we have some nice software for colocalization.
Choose products and then colocalization.
On Thu, 18 May 2000 11:16:43 +0200, Cornelius Krasel
<krasel at wpxx02.toxi.uni-wuerzburg.de> wrote:
>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.