Dear Flow-people, REcently I started to perform indirect antobody staining for protein which has nuclear localization. I checked this MoAb by doing immunofluorescense (fixation was done by 3% pharmaldegade and then I blocked nonspecific staining by 4% non-fat milk). Under the microscope my negative and positive cell lines look perfect. Althouth for FACS I used different protocol for fixation (80% iced-cold EtOH, ON), I didn't expect problems. So by flow I don't see the difference between negative and positive cell lines. Am i loosing my protein or I is it nonspecific staining? Could anyone share any experience with me? I'll appreciate any advises about fixation protocol, blocking, staining nuclear proteins. Thank you. 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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