Immunofluorescence analysis

Immunofluorescence on human testis sections (n=4) was performed after deparaffinization and rehydration by decreasing alcohol grades. Antigen retrieval was performed microwaving sections immersed in 10mM Sodium Citrate Buffer (pH 6.0). Permeabilization was performed with 0.1% Triton v/v for 10 minutes and blocking was achieved with 5% Donkey Serum-PBS v/v solution for 30 minutes (Sigma Aldrich, MI, USA). Sections were incubated with primary antibodies overnight at 4°C: anti-PDE8A (Atlas Antibodies Cat#HPA007722, RRID : AB_1855130), PDE8B (Atlas Antibodies Cat#HPA036912, RRID : AB_10670377), anti-ACROSIN (Biosonda, Santiago, Chile; Cat No. AMC-ACRO-C5F10-AS), Anti-Golgin-97 (SC-59820, Santa Cruz Biotechnology), anti-GM130 (610822 BD Biosciences), Lectin-PNA Alexa Fluor 488 ({"type":"entrez-nucleotide","attrs":{"text":"L21409","term_id":"424530","term_text":"L21409"}}L21409 ThermoFisher Scientific). After overnight incubation at 4°C, sections were washed in PBS and incubated with Alexa Fluor 488-Donkey anti-Rabbit IgG, Alexa Fluor 568-Donkey anti-Mouse IgG or Alexa Fluor 568-Donkey anti-Goat IgG antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for 1h at room temperature. Following extensive washes in PBS, sections were counterstained with DAPI and mounted with VECTASHIELD^® Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA). Confocal images were acquired as z-stacks on Zeiss Airyscan 2 with a 40x immersion oil objective (Carl Zeiss, Oberkochen, Germany). The acquisition was performed for 10 z-stack with a z-step of 0.5 mm and a frame size of 1024x1024 pixel. Pachytene spermatocytes and spermatids were identified by nuclear morphology as previously described (18, 19).