STED Microscopy of Sperm Actin Cytoskeleton

Epididymal sperm cells after swim out were diluted into PBS (1:10); 400 μl of diluted sperm were loaded on poly-L-lysine (CAS 25988-63-0, Sigma Aldrich)-coated coverslips in a six-well plate and air dried for 30 min at 37°C. After removing the PBS, sperm cells were fixed in 4% PFA followed by quenching with 50 mM NH₄Cl for 15 min. Sperm cells were permeabilized by 0.02% Triton X-100 for 3 min followed by washing with PBS, incubation for 1 h at RT with Phalloidin ATTO647 (1/1,000 in 3% BSA, ab176759, Abcam), and mounting with ProLong Gold Antifade (#P36930, Life Technologies). STED micrographs were acquired using a four-channel easy3D super-resolution STED optics module (Abberior Instruments, Göttingen, Germany) coupled with an Olympus IX73 confocal microscope (Olympus, Tokyo, Japan) and equipped with an UPlanSApo × 100 (1.4 NA) objective (Olympus, Tokyo, Japan) (Mikuličić et al., 2019 (link)), available in the LIMES Imaging Facility.