Vectashield anti bleaching solution

Cells were washed in PBS containing 1 mg/ml bovine serum albumin (BSA), and fixed in 3.7% paraformaldehyde (PFA) for 40 min at room temperature (RT) and then were permeabilised with 0.5% Triton X-100 for 30 min at RT. After washing, cells were further incubated at 4°C over night with antibody that recognized FLAG (5 μg/ml, Sigma Aldrich). Secondly, AlexaFluor 488- conjugated antibody (Life Technology) was diluted 1: 1000, in which the cells were incubated for 1 h in the dark at RT. After washed three times, cells were mounted in 3 mg/ml 4,6- diamidino-2-phenylindole (DAPI; Sigma aldrich) for 25 min at RT. And then, cells were moved into 20 μl 2% Vectashield anti-bleaching solution (Vector Laboratories). Fluorescence was captured by Carl Zeiss LSM 700 and saved in TIFF format. Besides, cells were transfected with pEGFP-N1 were not fixed and permeabilised but directed mounted in 10 μg/ml Hoechst 33342 (Sigma) for 15 min at room temperature. Then, fluorescence was detected at 10 × 40 using an inverted microscope (IX71, Olympus).

Oocytes and embryos were fixed with 3.7% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min at room temperature. After being washed with PBS containing 1% BSA (1% BSA/PBS), the cells were permeabilized with 0.2% Triton X-100 in PBS for 30 min and then washed with PBS containing 0.05% Tween 20. The cells were incubated with 4 N HCl containing 0.1% Triton X-100 for 10 min at room temperature and then transferred into 0.1 M Tris-HCl (pH 8.5) containing 0.02% Triton X-100. After a 30-min incubation, the cells were washed in 1% BSA/PBS and then incubated with an antibody against H2Bub1 (Medimabs, Mont-Royal, QC, Canada; #MM-0029-p; 1:200 dilution with 1% BSA/PBS) overnight at 4 C. After washing with 1% BSA/PBS, cells were incubated with a fluorescent-labeled secondary antibody, namely, Alexa Fluor 488 conjugated anti-mouse IgG (Life Technologies, Carlsbad, CA, USA, #A11001; 1:100 dilution). Cells were mounted on a glass slide in Vectashield antibleaching
solution (Vector Laboratories, Burlingame, CA, USA) containing 3 μg/ml 4’,6-diamino-2-phenylindole (DAPI; Dojindo, Kumamoto). The fluorescence signals were detected using an LSM 5 Exciter confocal microscope system (Carl Zeiss, Oberkochen, Germany).