I have starting doing some immunofluorescent staining and my cells are staining but are very dim. If anyone has some tips to improve staining I would appreciate it. Here is the general method I am using, I have tried a lot of variable already and have tried titrating both primary and secondary antibodies. I am staining adherent cells. Wash cells with PBS + 1% BSA Fix with 4% paraformaldehyde for 20 minutes at RT Wash with PBS + 1% BSA Permeabolize with PBS + 1%BSA + 1% Tween-20 for 6 minutes at RT Wash 3X with PBS + 1%BSA Stain primary antibody (mouse IgG) in PBS+1%BSA+0.1%Tween-20 for 1hr (up to O/N) at RT Wash 3X with PBS + 1%BSA Stain secondary antibody in PBS+1%BSA+0.1%Tween-20 for A) 45-60 minutes at 4C or B) 30-60 minutes at RT in the dark Wash 3X with PBS + 1% BSA. View on scope. Also, I know that there are some products out there that help the fluorochromes resist fading for a while, but can't remember the name or company that makes them. Any help on that appreciated as well. Thanks, BrianReceived on Fri Oct 22 14:58:00 2004
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