Rabbit anti sox2

Standard western blotting procedures were performed as described by Henderson et al.^{20 (link)}. The following antibodies were used: rabbit anti-CD51 (Abcam, Cambridge, MA, #112487, 1:1000); rabbit anti-p53 (Cell Signaling Technology, MA, #2527, 1:1000); rabbit anti-GAPDH (Cell Signaling Technology, MA, #2118 S, 1:1000); mouse anti-SP1 (Santa Cruz Biotechnology, CA, #sc-420, 1:500); mouse anti-SP3 (Santa Cruz Biotechnology, CA, #sc-136479, 1:500); mouse anti-Nestin (BD Biosciences, #611659, 1:1000); rabbit anti-Sox2 (Abcam, Cambridge, MA, #ab97959, 1:500)

Immunofluorescent staining was performed on 4% paraformaldehyde (PFA)-fixed cells as described by our research group previously [25 (link)]. The following primary antibodies were used in phosphate-buffered saline with 0.1% Tween detergent (PBS-T-0.1%)/ Triton X-100: mouse anti-rat Nestin 1:1000 (Sigma, Gillingham, Dorset, UK), mouse anti-GFAP (glial fibrillary acid protein) 1:1000 (Sigma, Gillingham, Dorset, UK), rabbit anti-GFAP 1:1000 (Agilent Dako, Cheshire, UK), mouse anti-Tuj1 1:1000 (Sigma, Gillingham, Dorset, UK), rabbit anti-TuJ1 1:1000 (Abcam, Cambridge, UK), mouse anti-RAGE receptor (Abcam, Cambridge, UK), anti-IB4 (isolectin B4) (Thermo Fisher Scientific, Paisley, UK), anti-HMGB1 (Abcam, Cambridge, UK) and rabbit anti Sox2 1:500 (Abcam, Cambridge, UK). Primary antibodies were probed using Alexa Fluor^® 488 or Alexa Fluor^® 555-conjugated anti-rat 1:1000, anti-mouse 1:1000, anti-Goat 1:1000 and/or anti-rabbit 1:500 secondary antibodies (Thermo Fisher Scientific, Paisley, UK). Cells were then counterstained with the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI; 5 µg/mL) (Sigma, Gillingham, Dorset, UK).

For immunofluorescence studies, the cells were grown on sterile coverslips in 24-well plates. On specified days, the cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 20 minutes, permeabilized with 0.1% Triton X-100 for 30 min and then blocked with 5% FBS in PBS for 30 min at room temperature. The fixed cells were incubated with mouse anti-MAP2 (1∶500; Abcam, Cambridge, UK), mouse anti-NeuN (1∶1000; Abcam, Cambridge, UK), rabbit anti-Sox2 (1∶250; Abcam, Cambridge, UK), rabbit anti-CD11b (1∶500; Abcam, Cambridge, UK), rabbit anti-GFAP (1∶500; Abcam, Cambridge, UK) and mouse IgM anti-O4 (1∶100; Sigma, USA) primary antibodies for 1 hr. The cells were then incubated with FITC-, Texas Red- and Cy5-coupled secondary antibodies (1∶200; Abcam, Cambridge, UK) for 1 hr. Hoechst 33342 was used as a nuclear stain. Images were viewed and analyzed using LSM710 confocal imaging software (Carl Zeiss MicroImaging Inc, Germany).

Cells or tissues were fixed, washed, and permeabelized with 0.1 % Triton X-100 for 30 min and then exposed to the following primary antibodies: mouse anti-Tuj1 (1:100, Covance, Dedham, MA, USA), mouse anti-Hu C/D (1:100, Life Technologies, Carlsbad, CA, USA), rabbit anti-p75 (1:500, Promega, Madison, WI, USA), rabbit anti-Sox2 (1:50, Abcam, Cambridge, MA, USA), rabbit anti-Doublecortin (1:250, Abcam), rabbit anti-Olig2 (1:250, Millipore, Billerica, MA), goat anti-GFAP (1:500, Abcam), goat anti-GFP (1:400, Rockland, Limerick, PA, USA). Secondary antibodies included goat anti-mouse IgG Alexa Fluor 546, goat anti-rabbit Alexa Fluor 488, donkey anti-goat Alexa Fluor 488, donkey anti-goat Alexa Fluor 546, donkey anti-mouse Alexa Fluor 546, donkey anti-mouse Alexa Fluor 488, all from Life Technologies (Carlsbad, CA, USA). Cell nuclei were stained with DAPI (Vector Labs, Burlingame, CA, USA).

H9‐ESCs seeded on the 1% Matrigel‐coated round coverslip in 24‐well plate on day 4 were fixed with 4% paraformaldehyde (PFA, Servicebio, Wuhan, Hubei, China) for 30 min at room temperature. After treating with 0.2% Triton X‐100 and 10% donkey serum and incubating at room temperature for 30 min, cells were incubated with the following primary antibodies in 10% donkey serum overnight at 4 °C: rabbit anti‐NANOG (1/200, Abcam, UK), rabbit anti‐OCT4 (1 : 250, Abcam) and rabbit anti‐SOX2 (1/200, Abcam). Subsequently, cells were incubated for 1 h with AlexaFluor‐568‐conjugated secondary antibody (1 : 400, Thermo) in 0.01 m phosphate‐buffered saline (PBS, Gibco, Grand Island, NY, USA) at room temperature. After washing cells with PBS for three times (5 min), the nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, 1 : 500, Sigma, Saint Louis, MO, USA) in PBS for 10 min at room temperature. Finally, images were captured at 200× magnification using a Zeiss Axio Imager Z2 microscope (Zeiss, Oberkochen, Germany) that was equipped with tissuefaxs software (TissueGnostics GmbH, Vienna, Austria) and prepared using imagej software (Version 1.52a, NIH, Bethesda, MD, USA) and prism 8 for macOS (Version 8.4.0, GraphPad Software, San Diego, CA, USA).

Primary antibodies used in this study included rabbit anti-SOX-2 (1:1000 dilution, Abcam, Cambridge, UK), rabbit anti-Nanog (1:1000 dilution, CST, Danvers, Massachusetts, USA), rabbit anti-OCT-4 (1:1000 dilution, Abcam), rabbit anti-pSmad2/3 (1:500 dilution, Abcam), rabbit anti-Snail (1:500 dilution, Abcam), rabbit anti-E-cadherin (1:500 dilution, Abcam), rabbit anti-TGF-β1(1:1000 dilution, Abcam), rabbit anti-N-cadherin (1:1000 dilution, Wanleibio), rabbit anti-Slug (1:1000 dilution, Wanleibio) and mouse anti-β-actin (1:1000 dilution, ZSGB-BIO, Beijing, China).