I am using CY-3 dye in my Confocal experiment. The florescence is relatively weak and it gets quenched almost instantly during scanning and acquiring the image. I m not sure if the problem is with the dye or the antifade mountant I am using. Dapi is fine however, therefore I think its the CY-3 which is problematic.
I'm not sure what your mountant is, but you could try a simpler mounting medium consisting of 90% glycerol with a bit of NaN3 and propyl gallate. I've yet to have much appreciable photobleaching on any of my ICC samples.
Thanks Jordan. Will give that a try. I am also trying a different Fluorophore just to be sure.
I suggest switching to Alexa dyes Alexa 568 or 594. The life technologies Prolong gold or diamond media is pretty goo dat preventing quenching. Alternatively Jordan's suggestion above is a very cost effective one!
Are you imaging DAPI before Cy3? You should do Cy3 first as imaging DAPI first can quench the Cy3 dye.
Thanks all. The problem has been rectified. The dye I was using was old and thus not the best.
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