Anti oct3 4 ab

Cells were lysed with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF and protease inhibitors). Protein samples were separated on an SDS polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore). Membranes were blocked using 1% or 5% BSA and incubated with the following primary antibodies: anti-p-ERK1/2 Ab (Cell Signaling; #9101L; 1:1000), anti-ERK1/2 Ab (Cell Signaling; #9102L; 1:1000), anti-Oct3/4 Ab (Santa Cruz Biotechnology; sc-5279; 1:1000), anti-Nanog Ab (ReproCELL; RCAB001P; 1:1000), anti-Otx2 Ab (Millipore; AB9566; 1:1000), anti-H3 Ab (Cell Signaling; #9715; 1:1000), anti-H3K27me3 Ab (Cell Signaling; #9733; 1:1000) or anti-Gapdh Ab (Santa Cruz Biotechnology; sc-32233; 1:1000). Membranes were then incubated with anti-rabbit IgG (Cell Signaling; #7074; 1:10,000) or anti-mouse IgG (Cell Signaling; #7076; 1:10,000) HRP-conjugated secondary Abs. Membranes were then washed and developed with ECL Plus reagents (GE Healthcare).

Cells were fixed with 4% paraformaldehyde in PBS and washed with PBS. Fixed cells were blocked with 1% BSA/0.3% Triton X-100 in PBS. For primary labeling, cells were incubated with anti-Oct3/4 Ab (Santa Cruz Biotechnology; sc-5279; 1:100), anti-Nanog Ab (ReproCELL; RCAB001P; 1:100) or anti-Otx2 Ab (Millipore; AB9566; 1:100). Later, the cells were stained with anti-mouse IgG Alexa Fluor 488-conjugated (Life Technologies; A-11001; 1:300) or anti-rabbit IgG Alexa Fluor 488-conjugated (Life Technologies; A-11008; 1:300) and Hoechst 33342 (Invitrogen; H3570; 5 μg/mL). Images were obtained using an LSM 700 confocal laser microscope (Carl Zeiss).