eGFP bleaching after immunostaining, desperately seek some help! - (Jan/18/2012 )

Hi everyone,

I use mammalian cell line U2OS for fluorescence microscopy experiments. I transfect the cells, 24hours later I check the egfp expression and then I fix the cell with fresh 3% PFA pH=7,4. I add antibody against different cellular proteins to the fixed cell, as a moulting medium I use the Invitrogene Pro Long gold antifade medium and I store the samples in the dark even through fixation and immunostaining procedure and still...some of the fixed samples show no signal after fixation. Things got even more strange last week, when I fixed eleven samples, checked all of them for the egfp expression before the fixation and after after the fixation and immunostaining, only four samples showed egfp expression. The rest of the cell looked just like untransfected control, even I used the same buffers and chemicals for all the samples and did the same methodics in the same day with all of them.

I know it sound crazy...but make this methodics work is crucially important for my PhD. I welcome any advice, what could possibly cause eGFP bleaching after immunostaining. Thanks a lot!

The fixation may be quenching the GFP fluorescence. You should be able to rescue the GFP signal by immunostaining for GFP.