Capacitation has been associated with changes in the lipid composition of the sperm plasma membrane; in particular, cholesterol efflux induces signaling events in the sperm, including the capacitation-associated increase in tyrosine phosphorylation and the preparation of the sperm to undergo the acrosome reaction [1, 5] . Previous work from our group has used a proteomic analysis of low-density DRM-resident proteins in an effort to investigate whether cholesterol efflux regulates membrane rafts during sperm capacitation [20] . The use of detergent to relate protein association to rafts is the most widely used method to analyze the composition of membrane rafts and to identify putative resident proteins [9] [10] [11] [12] [13] [14] . However, this procedure has been questioned on the basis that detergent can induce the formation of membrane domains and fails to provide physiologically relevant information [8, 14] . Despite these criticisms, detergent separation can still be considered a first approach to determining components of membrane rafts. In particular, although proteins not belonging to rafts could be found in the low-density DRM fractions, it is FIG. 5. Differential immunofluorescence pattern of GM3, CAV2, IZUMO, and FLOT2 in capacitated sperm. Mouse sperm incubated under capacitating conditions were analyzed by immunofluorescence as described in Materials and Methods using anti-GM3, anti-CAV2, anti-IZUMO, and anti-FLOT2, followed by the respective Alexa Fluor 555-conjugated secondary antibody. Cells were also stained with Alexa Fluor 488-conjugated PNA to relate the staining patterns to the acrosomal status. Sperm that underwent the acrosome reaction correspond to those showing the new staining patterns (arrowheads). Experiments were repeated at least three times; representative sperm are shown. DIC, differential interference contrast. Original magnification 360.