Immunol fluorescence staining secondary antibody dilution buffer

Twenty oocytes in each group were fixed with Immunol Staining Fix Solution (P0098, Beyotime, Shanghai, China) for 15 min at room temperature. They were then permeabilized with 1% TritonX-100 (P0096, Beyotime, Shanghai, China) for 20 min and incubated with immunofluorescence-blocking solution (P0228, Beyotime, Shanghai, China) for 30 min. The oocytes were subsequently incubated with Caspase-3 (A0214, Abclonal, Wuhan, China) antibody, diluted with QuickBlock™ Immunostaining Primary Antibody Dilution Solution (P0262, Beyotime, Shanghai, China), at 1:100, for 12 h at 4 °C. The oocytes were incubated with goat anti-rabbit IgG(H + L) (A0214, Beyotime, Shanghai, China), diluted with Immunol Fluorescence Staining Secondary Antibody Dilution Buffer (P0108, Beyotime, Shanghai, China), at a 1:200 ratio for 1 h at room temperature. After incubation, the oocytes were stained with Hochest 33342 for 10 min, and then washed with DPBS 3 times. The samples were observed under a Nikon eclipse Ti-S microscope (ti-2U, Nikon, Tokyo, Japan).

MG63 cells were washed three times with PBS and fixed with Immunol Staining Fix Solution (Beyotime) for 10 min, after which time they were washed with Immunol Staining Wash Buffer (Beyotime) 3 times for 5 min each. Actin-Tracker Green was diluted in Immunol Fluorescence Staining Secondary Antibody Dilution Buffer (Beyotime) and incubated with the cells for 40 min in the dark, after which they were washed with Immunol Staining Wash Buffer 3 times for 5 min each. Finally, nuclei were stained with UltraCruz^® Hard-set Mounting Medium including 4′,6-diamidino-2-phenylindole (DAPI; sc-359850, Santa Cruz Biotechnology). Images were recorded using a Zeiss confocal laser-scanning microscope equipped with a Plan-Apochromat 60× oil-immersion objective lens (Zeiss LSM 880 Confocal Laser Scanning unit, Carl Zeiss AG, Oberkochen, Germany). Image processing, combining and analysis were performed using the ZEN software package (Carl Zeiss MicroImaging GmbH, Jena, Germany).