Cy3 conjugated secondary antibody

Denuded oocytes were fixed and permeabilized using 1% paraformaldehyde, 0.2% Triton X-100 in phosphate buffered saline (PBS) for 1 h at room temperature, and then blocked in 3% bovine serum albumin (BSA) in PBS for 1 h. Incubation with first antibodies against ac4C (1:200, abcam, Cambridge, UK; Cat# ab252215) or NAT10 (1:200, ProteinTech, Wuhan, China; Cat# 13365-1) overnight was performed at 4°C, followed by three washes (5min each time) with 0.3% BSA. Incubation with cy3-conjugated secondary antibody (1:500, Earthox, CA, United States; E031620) was performed for 1 h at room temperature out of light, also followed by three washes with 0.3% BSA. Images were taken under an inverted fluorescence microscope IX73 (Olympus, Tokyo, Japan).

The oocytes were fixed in 1% paraformaldehyde and 0.2% Triton X-100 in PBS for 1 h at room temperature. After 1 h, the oocytes were transferred into 3% bovine serum albumin (BSA) in PBS to be blocked for another 1 h. Next, the oocytes were incubated with fluorescent-labeled antibodies against OGA (1:200), NAT10 (1:200), O-GlcNAc Transferase (OGT) (1:200), and/or another first antibody RL2 (1:200, Abcam, ab2739) at 4°C overnight. After three washes with 0.3% BSA, oocytes incubated with RL2 first antibody were then incubated with Cy3-conjugated secondary antibody (1:500, Earthox, E031620) at room temperature for 1 h in the dark condition. The oocytes were then washed with 0.3% BSA for three times. In addition, images were taken under the inverted phase contrast confocal microscope (LSM 880, Zeiss, JENA, Germany).