In contrast to what I might have implied in my earlier message; please don't use acetone fixed preps for confocal microscopy. Use formaldehyde fixed slides. It's no problem to se the acetone fixed slides to use in 'regular' fluorescence microscopy. Joost -----Original Message----- Dear Lars, in my opinion this is not a question to answer by flow cytometry, but use laser confocal microscopy. That way the exact localization of the protein can be visualized. An you are right, this will take fixation an permeabilisation of the cells. My suggestion would be the preparation of cytospins and fix (and perm) by acetone fixation. There is lots of literature dealing with this question this way. It is possible to use a flow cytometer to evaluate the total levels in the lymphocyte or (isolated) nuclei. See Purdue archive. Joost Schuitemaker Cell Biology and Histology Academic Medical Center Amsterdam http://www.amc.nl http://home.wanadoo.nl/flowcytometry -----Original Message----- From: Lars Brichta [mailto:LBrichta@gmx.net] Sent: maandag 23 september 2002 19:56 To: cyto-inbox Subject: [localization within lymphocytes] Dear all, is it possible to determine a protein in lymphocytes (isolated from whole blood) which is localized in the cytoplasmatic space and the nucleus as well? I think probably it would be necessary to fix the cells and to use a permeabilization agent which acts on both the cell membrane and the nuclear membrane as well? Does anybody know if there is a paper which gives any hints for such a method or the use of a kit? Thanks a lot! Lars Brichta Institute of Human Genetics University Clinics Bonn, Germany -- Werden Sie mit uns zum "OnlineStar 2002"! Jetzt GMX wählen - und tolle Preise absahnen! http://www.onlinestar.de
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