---------- Forwarded message ---------- Date: Sun, 2 Mar 1997 00:07:05 -0500 (EST) From: William George Telford <apoptosi@umich.edu> To: cyto-inbox Subject: Re: BrdU Staining Dear Paul, You might want to try a protocol described in Carayon and Boyd (J. Immuno. Meth. 147, 225-230, 1992), which uses a gentle fixation with paraformaldehyde and permeablization with Tween 20 followed by DNase treatment. The method has worked very well in our hands for phenotype-specific measurement of BrdU incorporation in mouse T splenocytes labeled for CD3, CD4, etc. No heating or acid-denaturation is used, which preserves surface marker expression and prevents the resulting cell damage and descrease in yield sometimes seen in some cell types. We immunophenotype prior to paraformaldehyde fixation and permeablization. Bill Telford Department of Pathology University of Michigan Medical School On Thu, 27 Feb 1997, Paul Zimmer wrote: > > Our laboratory has been using the standard acid-denaturation protocol > for testing BrdU incorporation by flow, but we have been disappointed by > the number of common epitopes destroyed during this process (e.g. CD23 > on murine B-cells). We have been testing the DNAse protocol described > by John Sprent's group, but with little success. All of the key > reagents were recently purchased and work well in other assays. I am > curious about two aspects of the protocol: > > 1) Does azide interfere with the DNAse enzyme activity? (we use azide in > our common stock of 1% paraformaldehyde solution) > > 2) Does the source of DNAse make a difference? (we have been using > molecular biology grade RNAse-free DNAse I) > > Any other suggestions or helpful comments on common problems would be > greatly appreciated. We have already spoken with Dr. Sprent's lab and > they have not had similar problems. > > -------- > J. Paul Zimmer, Ph.D. > Developmental and Clinical Immunology > University of Alabama at Birmingham >
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:49:28 EST