Alexa fluor 488 anti mouse igg

Immunofluorescence staining of sperm samples was performed as previously described [20 (link)]. Briefly, sperm samples were washed twice with phosphate buffer saline (PBS), fixed in 4% paraformaldehyde (Sigma-Aldrich, Castle Hill, NSW, Australia) at 4 °C overnight, and mounted on slides pre-treated with poly-L-lysine (Sigma-Aldrich, Castle Hill, NSW, Australia). Slides were incubated with primary antibodies (SLO3 (1:200, ABclonal Biotechnology, China), PLC-ζ1 (1:100, pab0367-P, covalab, USA), LRRC52 (1:500, PA5–107159, Invitrogen, Carlsbad, USA), CatSper1 (1:200; DF9349, Affinity Biosciences, Beijing, China), HSP60 (1:500; ab13532, Abcam, Cambridge, UK), acetylated alpha-tubulin (1:1000, mAb#5335, Cell Signaling Technology, Massachusetts, USA)) and PNA (1:500, RL-1072, VectorLabs, California, USA) overnight at 4 °C. After washing with PBS, slides were incubated with highly cross-adsorbed secondary Alexa Fluor 488 anti-mouse IgG (1:500, 34106ES60, Yeasen Biotechnology, USA) and Alexa Fluor 594 anti-rabbit IgG antibodies (1:500, 111–585-003, Jackson ImmunoResearch Inc., USA) for 1 h at 37 °C and subjected to Hoechst (1:1000, 62,249, Thermo Fisher Scientific Inc., USA) nuclear labelling for 2 h at 37 °C. Images were captured using an LSM 800 confocal microscope (CarlZeiss AG, Germany).

HCC tissue was fixed in 4% paraformaldehyde at 4 °C and then embedded in optimal cutting temperature compound and sectioned (20-μm thick) using a freezing microtome (Leica, Witzlar, Germany). After antigen retrieval, sections were blocked in phosphate-buffered saline supplemented with 5% bovine serum albumin for 1 h. Sections were then incubated with primary antibodies overnight at 4 °C. Secondary antibodies were Alexa Fluor 594-conjugated anti-rabbit IgG (1:200, Yeasen, Shanghai, China) or Alexa Fluor 488 anti-mouse IgG (1:200, Yeasen, Shanghai, China). Slides were exposed to DAPI nuclear stain (1:200, Yeasen, Shanghai, China) for 20 min, before being sealed with antifade mounting medium (Yeasen, Shanghai, China) and glass coverslips. Images were captured using a fluorescence microscope (Leica, Witzlar, Germany).