Egr1 antibody

The cells were seeded onto slides covered with 24-well plates and treated as indicated. The slides were incubated overnight with anti-rabbit Egr1 antibody (Cell Signaling Technology Cat# 4154) at 4°C, followed by incubation with a fluorescent secondary antibody at room temperature for 1 h. The nuclei were stained with DAPI for 15 min at room temperature. The images were captured using a fluorescence microscope (Zeiss, Gruppe, Germany).

The fixed tissue sections were deparaffinized, rehydrated, and antigen-retrieved in sodium citrate buffer (10 mM, pH 6.0) for 20 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide, and nonspecific binding was blocked with 10% normal horse serum. The sections were incubated with a primary antibody against Ly-6B.2 antibody (Bio-Rad, Hercules, CA, USA) or Egr-1 antibody (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, and then with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were incubated in an avidin–biotin–peroxidase complex solution (ABC solution; Vector Laboratories) for 30 min, and developed using a 3,3′-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories). Then, the sections were counterstained with hematoxylin, and the images were obtained using a CKX41 light microscope (Olympus).