Triton x 100 x100

Cultured cells were fixed with 4 % paraformaldehyde for 15 min, washed twice with PBS, and subsequently permeabilized with 0.5 % Triton X-100 (X100; Sigma-Aldrich) for 10 min. The washed cells were blocked for 30 min at room temperature with 1 % BSA (A9418; Sigma-Aldrich), and then incubated overnight at 4 °C with primary monoclonal mouse anti-alpha-actinin (A7811; Sigma-Aldrich) antibody diluted in 0.2 % BSA solution. After three serial washes with PBS, cells were incubated with AlexaFluor 594 conjugated goat anti-mouse antibody for 1 h at room temperature, and then washed and incubated in 300 nM DAPI for 5 min for nuclear counterstain.

Flow cytometry analysis of piPSCs cell cycle was conducted as previously reported with modifications [33 (link)]. The piPSCs before and after synchronization were harvested and preplated on uncoated 4-well dishes (176740, Nunc, Denmark) for 30 min to remove the feeder cells. After washing in phosphate buffered saline (PBS) twice, the cells were fixed in pre-cooled 70% ethanol at 4°C overnight. Then, the cells were incubated in staining solution containing 0.1 mg/ml RNase A (27062, iNtRON Biotechnology, Republic of Korea), 50μg/ml propidium iodide (P4170, Sigma) and 0.05% Triton X-100 (X100, Sigma) at 37°C for 40 min. Subsequently, the cells were resuspended in 500 μl PBS. Cell cycle analysis was performed on at least 3 independent cell samples. For each cell population, at least 10,000 cells were analyzed using a FACSCalibur Flow Cytometer (BD Biosciences), and the proportion in G0/G1, S and G2/M phases was estimated using the FlowJo V10 software (Tree Star Inc.).

Murine SPZ collected from caput and cauda epididymis were dried on slides as above reported and fixed in 4% paraformaldehyde (sc-281692; Santa Cruz Biotechnology, Heidelberg, Germany) for 20 min at RT and then permeabilized with 0.2% Triton X-100 (X100; Sigma-Aldrich, Milano, Italy). Blocking was carried out with 10% of donkey serum (ab7475; Abcam, Cambridge, UK) for 30 min at RT and then cells were separately incubated with different primary antibodies [IZUMO1 (ab211623) from Abcam Cambridge, UK; PNA (L21409) from Invitrogen, Milano, Italy; TSSK6 (sc-514076) from Santa Cruz Biotechnology, Heidelberg, Germany] overnight at 4°C. Following three washes in DPBS (1X), a fluorescein isothiocyanate (FITC) conjugated was used as secondary antibody (711-095-152; Jackson ImmunoResearch, Cambridge, UK) for 1 h at 37°C. Nuclei were labeled with DAPI (D9542; Sigma-Aldrich, Milano, Italy), while F-actin was labeled with phalloidin (21834; Thermo Fisher Scientific, USA). All samples were analyzed under an optical microscope (Leica DM 5000 B + CTR 5000) with a UV lamp. Densitometric analysis of immunofluorescence was performed with ImageJ Software (version 1.53 g) and adjusted relatively to DAPI fluorescence intensity.