Rabbit anti human nanog antibody

Mouse Anti-human CD24 antibody (Cat.: ab30350), rabbit anti-human CD44 antibody (Cat.: ab157107), rabbit anti-human CD133 antibody (Cat.: ab19898), rabbit anti-human Oct4 antibody (Cat.: ab19857), rabbit anti-human Nanog antibody (Cat.: ab21624), rabbit anti-IRE1 (phosphor S724) antibody (Cat.: EPR5253), rabbit anti-IRE1 antibody (Cat.: ab96481), rabbit anti-PERK (phosphor T982) antibody (Cat.: ab192591) and rabbit anti-PERK antibody (Cat.: ab65142) were obtained from Abcam (Cambridge, England) and diluted followed the manufacturer’s instructions.
Total protein was prepared using RIPA buffer (Thermo Scientific, Waltham, MA, USA) followed the manufacturer’s instruction. The same amount of protein from total protein was fractionated using Tris-glycine gels, and transferred to PVDF membranes. After transferring, PVDF membranes were blocked in 5% milk/TBS buffer at room temperature for 30 min, and this was followed by an incubation with primary antibodies at 4°C overnight. After washing with PBS-T (containing 0.1% Tween-20) for three times, HRP-conjugated secondary antibodies were incubated with PVDF membranes for another 1 hour. Blots were developed using Pierce^™ ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

Cells were placed on poly-L-lysine-coated glass coverslips and cultured in DMEM/F12 with 10% FBS at 37°C with 5% CO₂. The cells were fixed with 4% paraformaldehyde for 20 min and washed with PBS; 0.1% Triton X-100 was used to permeabilize the cells, and 0.5% BSA was used as a blocking agent. After washing with PBS, the cells were incubated with a rabbit anti-human Nanog antibody (1:500, Abcam, USA) overnight at 4°C. After washing with PBST, the cells were incubated with a secondary antibody (Alexa Fluor^® 488 Goat Anti-Rabbit IgG, 1:200, Invitrogen, USA) for 30 min; nuclei were stained with DAPI (CWBIO, China) for 5 min. The images were visualized by fluorescence microscopy (Olympus, Japan).