Sperm were fixed in suspension by the addition of fresh formaldehyde (prepared from paraformaldehyde [final concentration, 2%]) and incubation for 30 min at 48C. After washing two times with PBS, sperm were immobilized on slides and air dried. Cells were permeabilized with 0.1% Triton X-100 and 0.2% formaldehyde for 5 min at RT. After washing with PBS, blocking was done by 30min incubation with 1% BSA in PBS. Primary antibodies were diluted in PBS supplemented with 0.1% BSA and incubated overnight at 48C. After washing with PBS, cells were incubated with the respective Alexa Fluor-conjugated secondary antibody for 60 min at RT. When double staining was required, PNA coupled to Alexa Fluor 488 was included in the solution of the secondary antibody. After washing, cells were mounted using Slow Fade (Molecular Probes).