Em grade

Caudal epididymal spermatozoa were isolated in PBS from wild type (WT) and Ppp3r2 knockout mice and were centrifuged at 700×g for 10 min at 4°C. Spermatozoa were fixed in 4% paraformaldehyde, EM grade (Electron Microscopy Sciences) at 4°C for 20 min, followed by permeabilization with 0.2% Triton-X (5 min). Fixed spermatozoa were attached to poly-L-lysine coated coverslips. The coverslips were washed three times with TTBS to remove excess paraformaldehyde and incubated for 4 hours in a blocking solution containing 5% non-immune serum in TTBS at room temperature. The coverslips were incubated overnight at 4°C with one of the following primary antibodies: PPP3R2 antibody (1:150 dilution; Proteintech; Cat # 14005–1-AP), MCT2 (1:200 dilution; sc-166925), Basigin (1:200 dilution; Invitrogen; Cat # 12-1471-81), DRP1 (1:150; BD Biosciences; Cat# 611738) and p-DRP1 (1:150 dilution; Cell Signaling; Cat# 3455S). Coverslips were subsequently washed three times 5 min each with TTBS, followed by incubation with the appropriate secondary antibody conjugated to Cy3 or Alexa flour488 for 1 hour at room temperature. The coverslips were washed three times, 10 min each with TTBS; cells were then stained with Hoechst. Mounting medium was applied, and the slides were examined by confocal laser scanning microscope (Olympus IX81 attached with FLUOVIEW FV1000).

Cells were fixed in 4% paraformaldehyde (EM grade, Electron Microscopy Sciences) for 10 min, rinsed three times in PBS and permeabilized for 10 min in PBS/0.2% Triton X-100 at room temperature, followed by three rinses in PBS. After blocking for 30 min at room temperature in PBS/5% FBS, slides were incubated with the primary antibody against DDX20 (mouse monoclonal, sc-57007, Santa Cruz Biotechnology, 1:200 dilution) or SMN (rabbit polyclonal, sc-15320, Santa Cruz Biotechnology, 1:200 dilution) in PBS/5% FBS at 4°C overnight. After three rinses in PBS, slides were incubated with the secondary antibody (1:1,000 dilution in both cases) and Hoechst nuclear dye (1:10,000 dilution) for 90 min at room temperature. The secondary antibody for DDX20 was a goat anti-mouse Alexa 594 antibody (Invitrogen) and the secondary antibody for SMN was a donkey anti-rabbit Alexa 568 antibody (Invitrogen). Slides were rinsed three times in PBS and once in water. If no FISH was performed subsequently, slides were mounted.