Anti sox2 antibody

Reagents used for cell culture were purchased from GIBCO (Invitrogen Corporation, Carlsbad, CA). Anti-RAGE-blocking antibody used in animals was purchased from RD System (Minneapolis, MN). LPS was purchased from Sigma–Aldrich (St. Louis, MO, USA); recombinant HMGB1 protein was purchased from Novus Biologicals (Colorado, USA). Anti-nestin antibody was purchased from Cell Signaling (Danvers, MA). Anti-RAGE antibody, anti-MAP-2 antibody, anti-SOX-2 antibody, and Tnf-a and IL-1β ELISA kits were purchased from Abcam (Cambridge, UK).

GC cells that had reached 60%-70% confluence on coverslips in 12-well plates were fixed with paraformaldehyde (4%) for 15 min, followed by quenching of autofluorescence three times with 0.1 M glycine for 10 min each, permeabilized with Triton X-100 (0.4%; 15min), treated with acetic anhydride (0.25%) for 10 min, and hybridized with Cy3-labeled oligonucleotide probes in hybridization solution at 45°C for 16 h. GC cells were then blocked with BSA (1%; 37°C) for 1 h, incubated overnight (4 °C) with anti-SOX2 antibody (1:200, Abcam, Cambridge, UK), and with Cy3-labeled secondary antibody (2 h; 25°C). The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) and the cells were imaged using a TCS SPE confocal laser-scanning microscope (Leica, Mannheim, Germany).

The pathological sections of NSCLC patients’ tumor tissues were retrieved by microwave antigen retrieval (Citrate-EDTA Antigen Retrieval Solution, Beyotime). The sections were then blocked with 5% BSA and incubated with mouse monoclonal anti-integrin αvβ3 (1:100, ab7166, Abcam, UK), rabbit monoclonal SOX2 (1:200, ab92494, Abcam, Cambridge, UK), rabbit polyclonal p-AKT (1:200, ab38449, Abcam, Cambridge, UK), rabbit monoclonal KLF (1:200, ab215036, Abcam, Cambridge, UK), rabbit monoclonal total-PI3K (1:500, ab302958, Abcam, Cambridge, UK), mouse monoclonal CD90 (1:100, ab181496, Abcam, Cambridge, UK) for 4 °C overnight, and followed by horse radish peroxidase (HRP) conjugated secondary antibodies (1:600; Abcam, Cambridge, UK), and the nucleus was stained with DAPI. In addition, the A594 and Lewis cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% triton-X100, and blocked by 5%BSA, followed by incubating with anti-SOX2 antibody (1:200, Abcam, Cambridge, UK) for 4 °C overnight, and followed by HRP conjugated secondary antibodies (1:600; Abcam, Cambridge, UK), and the nucleus was stained with DAPI. All immunofluorescence images were captured from FV1000 laser scanning confocal microscope (Leica, Barnack, Germany) and analyzed by IPP software.

Cell samples were fixed in 4% (v/v) paraformaldehyde solution (MultiSciences Biotech, Hangzhou, China) at room temperature for 15 min. Endogenous peroxidase was inhibited by immersion in a 0.3% (v/v) hydrogen peroxide (H₂O₂) in methanol bath for 15 min. After washing, nonspecific binding was blocked with 5% (m/v) bovine serum albumin in PBS. The cells were incubated with diluted primary antibody Anti-SSEA4 antibody (15 μg/ml; Abcam), Anti-TRA-1-60 (R) antibody (1 : 100; Abcam), Anti-OCT4 antibody (5 μg/ml; Abcam), Anti-NANOG antibody (1 : 1000; Abcam), Anti-SOX2 antibody (1 μg/ml; Abcam), Anti-FABP4(1 : 1000; Abcam), Anti-PPAR gamma (5 μg/ml; Abcam), Anti-RUNX2 (1 : 500; Abcam), Anti-Osteocalcin (10 μg/ml; Abcam), Anti-human AFP (1 : 200; Abcam), Anti-human CK18 (2 mg/ml, 1 : 400; Abcam), Anti-human CK19 (1 : 400; Abcam). According to the manufacturer's instructions, detection of primary antibody was performed using horseradish peroxidase-conjugated secondary antibodies (1 mg/ml, 1 : 1000; Abcam). Peroxidase activity was revealed by a 3- to 5-min exposure to diaminobenzidine tetrahydrochloride solution (DAB kit, Vector Labs, Burlingame, CA, USA). The fixed cells were then washed, counterstained with hematoxylin for 1 min, mounted and observed under an inverted phase-contrast microscope (ECLIPAS TS100, Nikon, Tokyo, Japan).