Thanks to everyone for response to my letter one week ago. I decided to use 3 different protocols of fixation: -80% -20C ETOH for 30', then with or without blocking agent(5%non-fat milk) -70% -20C Methanol for 30', with or without blocking agent -3%formaldegade for 30' RT, with and without block; For all washing I used 1xPBS/0.01% Tween-20/0.1%BSA For MoAb staining I used 1xPBS/0.05%Tween/1% BSA Staining was done at RT for 1 hour for 1st and 2nd ab. As a control i used unstained cells and cells treated with 2nd ab only. Now I have really significant nonspesific staining, no matter did I block receptors or not. Actually it seems that milk didn't work here as a blocking agent. Is anyone could give me some advises how to get ride of nonspesific staining? Thank you all very much. Marina Dr. Marina G. Polonskaia Research Associate Harvard Medical School Flow Cytometry Facility tel: (617)432-3647 pager:(617)465-8982 e-mail: mpolonskaia@hms.harvard.edu
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