Hi Laura, You are right in saying that the acid treatment destroys surface staining so the DNAse method is the one to go for. I fix cells in 1% paraformaldehyde with 0.01% Tween, then after washing add 1ml of PBS (making sure that it contains Ca and Mg) and 50Kunitz DNase I (from bovime pancreas and Sigma). Leave for 30 mins at 37C, wash then do BrdU Ab staining. I find that I get a stronger BrdU signal with this method although, if you are doing DNA as well, the profile isnt as good as after ethanol fixation. 20000 Kunitz worth of DNase costs around 120 of your US$ and should do around 3-400 samples. Hope this helps and good luck! Derek On Tue, 30 Mar 1999, Laura Healy wrote: > I received a protocol (faxed from a comercial source) for surface labeling > and BrDU incorporation using FACS permeabilizing soln. and DNAse. I was > told the protocol came from the Purdue web site, but I can't find it. What > I need is what kind of DNAse should I get for this protocol? It requires > filtration according to the protocol. It also calls for a 10 mg/ml > solution of DNAse. That's about $50. worth for 1 ml (?)- and 1/2 is lost > in filter. Thanks for any responses! I've tried acid, but it destroys my > surface marker (PE). > > Laura Healy **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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