You could try staining with your surface ABs then fix with BD FACS Lyse, give the cells a wash or two then stain with your nuclear antibodies. This works well for Ki67 and BRDU while preserving surface staining, I dont see why it shouldn't work for you. You can further improve the nuclear staining by adding a tad of NP40 to your PBS. I could be more specific if you want or send you our new improved but tedious BRDU protocol. Best of luck Simon Simon Monard FACS Lab Manager Trudeau Institute Saranac Lake NY12983 Ph 518 891 3080 X352 >>> Calman Prussin <CPRUSSIN@niaid.nih.gov> - 4/18/02 7:33 PM >>> We are doing intranuclear staining for transcription factors, which requires a methanol fixation/permeabilization step. We are finding that the methanol fixation dramatically reduces APC fluorescence (of cell pre-stained with mAb). Staining protocol (simplified) 1. 4% PFA fix 2. stain for surface markers (FITC, PE, APC conjugated) 3. 80% methanol (-70 C) fixation 4. Perm with 0.5% saponin, stain for transcription factors with PE/Cy5 labeled secondary mAbs 5. Run We find that methanol fixation reduces or eliminates the APC fluorescence, but does not change the PE. Questions: 1. Has anyone else seen this? 2. Why doesn't it change the PE fluorescence? 3. Solutions? The only thing I can think of is to switch to Cy5.
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