Fig 2: Immunocytochemistry of homozygous SAO erythrocytes. Circulating erythrocytes from the homozygous SAO individual, containing both large, aberrantly-shaped homozygous SAO cells and normal biconcave donor cells, were imaged using immunofluorescent labeling and confocal microscopy. (A) Red cells were reacted with monoclonal antibodies directed to extracellular epitopes of membrane proteins of interest: Band 3 (BRAC17), RhAG (LA1818), GPA (BRIC256), GPB (and to a lesser extent GPA) (R1.3), Wr(b) (BIRMA 84b, BRIC13 and BRAC13), CD47 (BRIC211), CD147 (MEM6-6), CD44 (BRIC235), and GPC (BRIC10). (B) Red cells were co-stained with erythroid marker anti-GPB (and to a lesser extent GPA, R1.3; red) and DNA dye DAPI (blue). The white arrow indicates a leukocyte, the red arrow indicates an erythroid cell that has retained nuclear material. Numbers of nucleated erythroid cells were not sufficient to quantify accurately but were seen occasionally in the confocal slides of homozygous SAO peripheral blood whereas they are very rare in control slides. Images are representative of at least 5 different fields from one experiment. Scale bars represent 5 µm.