Hi, I want to do an intracellular and extracellular staining of Fas and I thought of doing it in the following way: First stain for extracellular Fas, fix the cells, permeabilise and stain for intracellular Fas using either PE/FITC conjugated antibody respectively. Is this a good way to do it? Also, I wanted to know the protocol to be followed for permeabilisation of cells as I am using brain tumor cells. Does the protocol differ for cell type? I have tried to permeabilise using either 70% ethanol, triton x-100 or saponin but I always tend to lose cells. I would like to know the speed of pelleting the cells and how one washes after permeabilisation as I lose cells at this stage. I appreciate a quick response for this mail Thank you Bharathi Bharathi L., Ph.D. Human Medical Genetics Unit A209 Medical Alumni Building University of Vermont College of Medicine Burlington VT 05405. USA Telephone Lab: 001 802 656 0390 Res: 001 802 658 1518 FAX: 001 802 656 2641 E-mail: blaxman@zoo.uvm.edu
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