Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole

HCT‐116 cells were grown on glass coverslips in multiwell plates, fixed with 4% paraformaldehyde for 10 min at 4 °C, and permeabilized with 0.1% Triton X‐100 for 10 min at room temperature. Cells were then washed with PBS; blocked in 1% bovine serum albumin, 0.1% Tween, and 2% glycine for 1 h at room temperature; and incubated with anti‐progranulin (1 : 100 final dilution, PA5‐27275) and anti‐p‐STAT3 Tyr705 (1 : 50 final dilution, sc‐8059) antibodies overnight at 4 °C. Subsequently, cells were rinsed with PBS and incubated with Alexa Fluor 546 (goat anti‐mouse) and Alexa Fluor 488 (donkey anti‐rabbit) (both from Life Technologies) for p‐STAT3 Tyr705 and progranulin detection, respectively, for 1 h at room temperature in the dark. After rinsing in PBS, cells were mounted using Prolong gold antifade reagent with 4′,6‐diamidino‐2‐phenylindole (Life Technologies) and visualized with a Zeiss LSM 800 Confocal Laser Scanning Microscope equipped with a 63 × objective (Carl Zeiss, Milan, Italy). Fluorescence images were processed using ZEN 2.6 (Blue Edition, Carl Zeiss).

For flow cytometry, sperm were suspended in prewarmed staining solution (0.1% bovine serum albumin in PBS) containing MitoSOX Red (Thermo Fisher Scientific). After incubation for 10 min at 37 °C and 5% CO₂, sperm were rinsed three times with prewarmed PBS and then analyzed with flow cytometry (CYTEK NL-3000). Data were analyzed with FlowJo version 10.8.1 (https://www.flowjo.com/solutions/flowjo/downloads/previous-versions).
For confocal microscopy, sperm were seeded on 8-well Chamber Slide (Nunc) with Cell-Tak and then centrifuged for 1 min at 700g. After incubation with MitoSOX Red for 10 min at 37 °C and 5% CO₂, sperm were washed with prewarmed PBS and then with PBS containing 4% formaldehyde for 30 min. After rinsing with PBS three times, the sperm were permeabilized with 0.1% Triton X-100 at room temperature for 20 min and washed three times with PBS. They were then incubated with Phalloidin-Green (Hello Bio) for 30 min, rinsed three times with PBS, and incubated with ProLong Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole (Life Technologies) for 2 h. Fluorescence localization was observed using a Leica Stellaris 8-STED Super-resolution Confocal Microscope (Leica). Image analysis for the fluorescence localization was performed using LAS AF Lite (Leica). Data were processed by CellProfiler and analyzed by GraphPad Prism software (https://www.graphpad.com/features).