Immunocytochemistry of red blood cells. Red blood cells from band 3 +/+ and band 3 −/− mice were fixed onto Alcian Blue-coated coverslips with 4% paraformaldehyde. After permeabilization of the cells in a PBS solution containing 0.1% Triton-X 100 and blocking in a PBS solution with 1.0% bovine serum albumin and 1.0% goat serum, the cells were incubated with a rabbit antibody raised against the cytoplasmic domain of human GPA. The signal was detected with a goat antirabbit/rhodamine-conjugated secondary antibody. In contrast to band 3 +/+ red blood cells, band 3 −/−red blood cells do not display any detectable level of GPA on their surface.