Mab 414 antibody

For IF staining of PBRM1, the fresh bull semen spread onto polylysine-coated slides was air dried, fixed using 4% paraformaldehyde for 5 min, washed three times in PBS, permeated in 0.2% Triton X-100 for 15 min, and blocked using 5% BSA for 1 h at room temperature. After washing with PBS three times, the samples were incubated with primary antibodies diluted with PBS (including anti-rabbit PBRM1 [1:100] (AB196022, Abcam, Cambridge, UK) or mAb 414 antibody (1:100, 902,097, BioLegend, San Diego, USA)) at 4°C overnight. Then, samples were incubated with secondary antibodies, including Alexa Flour® 488-conjugated goat anti-rabbit IgG (1:150; ZF-0511, ZSGB-BIO, Beijing, China) for 1.5 h at 37°C. For co-localization analysis of the nuclear pore complex (NPC) and PBRM1, the mAb 414 antibody was visualized by using a TRITC-conjugated secondary antibody, and PBRM1 labelling was visualized by using a FITC-conjugated secondary antibody. The samples were incubated in DAPI staining solution (C1005, Beyotime, China) for 10 min at room temperature and washed three times with PBS. The samples were added with an anti-fluorescence quencher and placed in a cover glass, and the edge was sealed with nail oil. Immunofluorescence staining was imaged at 200× magnification using an inverted fluorescent microscope (IX73, OLYMPUS, Japan).