Immunoflourescent Staining Help Needed

From: Brian Newsom <BrianN@OpexaPharma.com>
Date: Fri Oct 22 2004 - 12:03:34 EST
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,
Brian
Received on Fri Oct 22 14:58:00 2004

This archive was generated by hypermail 2.1.8 : Thu Oct 28 2004 - 03:12:04 EST