From: Peter Schroeder (peter.schroeder@medtronic.com)
Date: Tue May 12 1998 - 11:30:14 EST
Thanks for all the information on the CD62 and PFA fixing. From some of the responses I judged it would be helpful to post some of the info to the group. The take home message was that fixing with 1% paraformaldehyde after the staining with CD62 would give the same results as samples that were never fixed but labeled and run immediately (K.A. Ault, Biocytex). Thanks Again, Peter Schroeder Center for Biomaterials Research Medtronic, Inc. Kenneth A. Ault M.D. made this interesting comment on "pre-staining": You have to remember that although resting platelets have no P-Selectin ontheir surface, they have lots of it in their alpha granules. Thus, if the platelets become permeabilized so that the antibody can gain access to theinterior of the platelet, you get lots of staining. Heavy fixation causes some permeabilization, and I think this is what others have observed when theyreport that fixation "causes activation". Bioctytex sent me the following information regarding their platelet activation kit sold by Alexis: Paraformaldehyde fixation can be used in two different ways. First way : Sample fixation before staining to store the sample. In this case cell membranes can be altered and an increase of MAb binding can be observed. We have studied the influence of 1% PFA fixation on PRP and we have obtained the following results : PRP (n=30) 1% PFA fixed PRP* (n=8) CD61 51,000 +/- 9,000 70,000 +/- 8,800 CD62 < 300 100 to 700 CD42b 37,000 +/- 6,000 42,000 +/- 10,000 * PRP fixation with 1% PFA (vol/vol) Incubation 10 min. Two washes in PBS buffer (1,200 g - 10 min.). As Cahill reported, platelet fixation induces an increased expression of the antigen (I don't know if it is due to platelet activation or membrane alterations). Second way : Sample fixation after staining to differ cytometry analysis. In this case paraformaldehyde stabilizes MAb binding with no effect on Gp measure. "PLATELET Gp" procedure includes this final fixation step. We have some data to compare technical protocol including final fixation with technical protocol without this fixation step and we did not observe significant differences in quantitative values. Thus, don't be affraid to fix CD62 stained samples.
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