Histological analysis

For H&E staining, semen smears were dehydrated with graded ethanol, stained with H&E, dehydrated again with graded ethanol and processed with dimethylbenzene for 5 min twice. For histological analysis, human testicular tissue from a patient with prostate cancer with normal fertility were fixed in Bouin’s solution (Sigma-Aldrich), embedded in paraffin, sectioned, processed, saved and used for subsequent experiments. Immunofluorescence analysis was performed as previously described.¹⁹ The slides were incubated with primary antibodies (CFAP65, SPAG6 and anti-acetylated tubulin monoclonal antibody) for 2.5 hours at 37°C. The details of all antibodies are listed in online supplementary table S1. The slides were then incubated with secondary antibodies (Alexa Fluor 488 anti-mouse IgG (A-21121, 1:400) and Alexa Fluor 555 anti-rabbit IgG (A31572, 1:400)) for 1.5 hours at 37°C. For evidence of the acrosome, the samples were treated with Pisum sativum agglutinin (PSA) and incubated for 2.5 hours at 37°C. Finally, all slides were stained using 2-(4-amidinophenyl)-1H-indole-6-carboxamidine for 5 min at room temperature. An Olympus IX51 fluorescence microscope (Olympus, Tokyo, Japan) and VideoTesT-FISH 2.0 software (VideoTesT, Petersburg, Russia) were used for photographing fluorescence signals.

jmedgenet-2019-106031supp001.pdf