You might try our protocol using brief glutaraldehyde fixation and triton X permeabilization, which was published in Transfusion last year (Davis BH, Olsen S, Bigelow NC, Jenn JC: Detection of fetal red cells in fetomaternal hemorrhage using anti-hemoglobin F monoclonal antibody by flow cytometry. Transfusion, 38:749-756, 1998) and also detailed in the anti-HbF antibody sold by CALTAG laboratories. However, all our work was done on human blood, so I can not vouch with absolute certainity how it would work with mice, but in our hands the method is better (and faster) than formaldehyde. We also have recently devised a slick and easy method for counting F cells, so if the method works on mice cells and F cells is your interest let me know for more details. Good luck, Bruce H. Davis, M.D. Wm Beaumont Hospital Dana Levasseur wrote: > Hello Everyone, > > I am trying to find a protocol for fixing and permeabilizing mouse > erythrocytes to allow intracellular staining for fetal hemoglobin levels > with a HbF-FITC antibody. I can only assume that C57Bl/6 RBCs are > significantly more susceptible to hemolysis than human RBCs. People have > treated human RBCs for hours at room temp in 4% formaldehyde before > permeabilization with Triton. Mouse RBCs lyse after short duration > exposure to 4% formaldehyde at 4 degrees and a single pelleting at low > speed. Any ideas on how to handle such a fickle beast would be greatly > appreciated?!! > > Dana > Dana Levasseur > Dept. of Biochemistry and Medical Genetics > Bevill Biomedical Research Building, Rm 263 > University of Alabama at Birmingham > Birmingham, Alabama 35205 > > e-mail: dnl@uab.edu
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