> >Phillippe- >Attached is a protocol that I adapted for CD8 surface staining of T cells >back in 1997. I tried many combinations of paraformaldehyde/methanol or >EtOH at various concentrations. The protocol outlined gave me the best >compromise between maintenance of surface label with low G1/G0 cv's >following PI staining. The 0.25% paraformaldehyde fixation was necessary >to immobilize the surface antibody, but higher concentrations resulted in >high cv's - probably as a result of crosslinking nuclear proteins. The >methanol gave better results compared to ethanol. Regardless, both >reduced surface staining, to some extent. I found this method did not >work well for antibodies where the antigen is expressed at low levels >(i.e. CD3), but it worked well for CD8. > >Best wishes, >Julie > > >At 03:01 PM 1/22/02 +0000, you wrote: > >>Hi, >>We want to label CD8alpha expressing cells with an FITC conjugated Ab. >>This Ab works >>great on T cells (Pharmingen, clone >>53-6.7). But we need to have our cells EtOH fixed for further DNA content >>analysis >>with PI, instead of the usual formaldehyde >>procedure, which is incompatible with PI DNA content analysis. Up to now, >>our first >>tests are not very promising. Does anybody >>out there have some experience on that point, and good advice? >>Thank you in advance! >>Philippe >>PS: by the way, if someone knows where we can get the 53-6.7 clone, I am >>sure our lab >>budget would appreciate the tip... >> >>____________________________________________ >> >>Philippe Pognonec, Ph.D. >>Transcriptional Regulation and Differentiation >>Centre de Biochimie >>Parc Valrose >>Universite de Nice >>06108 Nice cedex 2 >>France >>Tel/fax: (33) 492 07 64 13 >>____________________________________________ > >******************************************************************************** >Julie A. Auger >Director, Office of Shared Research Facilities >University of Chicago, Biological Sciences Division >007 Kovler Lab >910 E. 58th Street >Chicago, IL 60637 > >e-mail: jauger@flowcity.bsd.uchicago.edu >telephone: 773-702-9212 lab 773-702-9261 office >fax: 773-702-7195 >************************************************************************
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