*2.5. Fluorescence Immunocytochemistry*

Mouse and boar spermatozoa were mounted on poly-L lysine coated coverslips and fixed in 2% formaldehyde for 40 min. Non-specific binding was blocked using 5% bovine serum albumin (BSA) for 25 min before incubation in primary antibody overnight at 4 ◦C. The next day, the coverslips were washed extensively in 1% BSA-PBS before a 40 min incubation with secondary antibody conjugated to a fluorescent marker and DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) at room temperature and hidden from light. The coverslips were washed again before being mounted on glass slides using VectaShield mounting medium (Vector Laboratories, Burlingame, CA, USA) and sealed with nail polish. Spermatozoa from mouse and boar were also incubated with our fluorescent inhibitor for 25 min, washed extensively and mounted onto glass slides to visualize the binding pattern. Fluorescence images were taken at the Queen's University Cancer Research Institute Imaging Centre, using a Quorum Wave Effects spinning disc confocal microscope or at the University of Missouri-Columbia, using a Nikon Eclipse 800 microscope with CoolSnap CCD camera (Nikon, Tokyo, Japan). All images were subsequently analyzed using MetaMorph Imaging Software (Molecular Devices, San Jose, CA, USA).
