Ec plan neofluar 63 1.25 objective

Cell slides or smears were prepared and then fixed with 4% paraformaldehyde for 20 min at 4 °C. Nonspecific binding sites were blocked with 10% BSA/PBS for 60 min at room temperature, followed with 0.1% TritonX-100 permeable treatment for 10 min. Sections were incubated with the TFEB antibodies (1:200 dilution; Santa cruz), GFRA1 antibody (1:200 dilution; Santa cruz) overnight at 4 °C. Then, fluorescence-labeled secondary antibodies (donkey anti–rabbit Alexa Fluor 488, donkey anti–mouse Alexa Fluor 555, 1:500 dilution; Jackson ImmunoResearch) were used. Nuclei were counterstained with DAPI (Sigma-Aldrich). The fluorescence signals were detected under a laser scanning confocal microscope (Carl Zeiss LSM-510, Germany) equipped with an argon laser (488 nm), a He/Ne laser (543 nm), an EC Plan-NEOFLUAR 63×/1.25 objective and a LD LCI Plan-APOCHROMAT 25×/0.8 objective (Zeiss). Digital images were taken and processed using Aim software (Zeiss Systems).

For IF staining, sperm smears or testicular sections were prepared on slides and C18–4 cells were cultured on glass-bottom dishes (Cellvis, Mountain View, CA, USA). Then, slides or dishes were fixed with 4% paraformaldehyde for 20 min at 4 °C. Nonspecific binding sites were blocked with 10% BSA/PBS for 60 min at room temperature, followed by 0.1% TritonX-100 permeable treatment for 10 min. Sections were incubated with the ZIP12 antibodies (Abcam, 1:200 dilution), and/or PLZF antibodies (Santa Cruz Biotechnology, 1:200 dilution, USA) overnight at 4 °C, respectively. Then, fluorescence-labeled secondary antibodies (donkey anti-rabbit Alexa Fluor 488 or donkey anti-mouse Alexa Fluor 555, 1:200 dilution; Jackson ImmunoResearch) were used. Nuclei were counterstained with DAPI (Sigma-Aldrich). The fluorescence signals were detected under a laser scanning confocal microscope (Carl Zeiss LSM-510, Germany) equipped with an argon laser (488 nm), a He/Ne laser (543 nm), an EC Plan-NEOFLUAR 63×/1.25 objective, and an LD LCI Plan-APOCHROMAT 25×/0.8 objective (Zeiss). Digital images were taken and processed using Aim software (Zeiss Systems) [30 (link)].