Plan apochrombat 63x 1

Mouse and human sperm were washed in PBS twice, attached on the glass coverslips, and fixed with 4% paraformaldehyde (PFA) in PBS at room temperature (RT) for 10 min (mouse) or at 4°C for 1 hr (human). Fixed samples were permeabilized using 0.1% Triton X-100 in PBS at RT for 10 min, washed in PBS, and blocked with 10% goat serum in PBS at RT for 1 hr. Cells were stained with antibody in PBS supplemented with 10% goat serum at 4°C overnight. After washing in PBS, the samples were incubated with goat anti-rabbit Alexa 647 or Alexa 555-plus (Invitrogen, 1:1,000) in 10% goat serum in PBS at RT for 1 hr. Hoechst was used to counterstain nucleus for sperm head. For BODIPY-N-ethylmaleimide labeling, reduced thiols within proteins were alkylated with BODIPY FL maleimide (final concentration of 10 nM, ThermoFisher) for 30 min in the dark after permeabilization. The sample was then quenched by the addition of 500 mM 2mercaptoethanol for 30 min in the dark, followed by 3 times washing in PBS. For ThiolTracker (ThermoFisher) labeling, fixed sperm were stained with 20 μM dye, followed by 3 times washing in PBS. Stained sperm samples were mounted with Prolong gold (Invitrogen) and cured for 24 hr, followed by imaging with Zeiss LSM710 Elyra P1 using Plan-Apochrombat 63X/1.40 or alpha Plan-APO 100X/1.46 oil objective lens (Carl Zeiss).