Sox2 antibody

To determine the changes in cytoskeleton structure and expression of different markers, CiSCs, BM-MSCs, and MCF7 cells were seeded on glass slides precoated with poly-d-lysine (Sigma-Aldrich, USA). Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 4% BSA. Cells were then stained with Alexa Fluor® 488 Phalloidin (Molecular Probes, USA), α-tubulin antibody (Cell Signaling Technology, USA), Ki-67 antibody (Cell Signaling Technology, USA), Oct-4 antibody (Cell Signaling Technology, USA), Sox2 antibody (R&D Systems, USA), Nanog antibody (Bioss Antibodies, USA), E-Cadherin antibody (Cell Signaling Technology, USA), N-Cadherin antibody (Abcam, USA), Snail + Slug antibody (Abcam, USA), ALDH1A1 antibody (Pierce Antibodies, USA), and β-Catenin antibody (Cell Signaling Technology, USA). Cells were labeled with the appropriate Alexa Fluor® secondary antibodies (Molecular Probes, USA) and counterstained with Hoechst 33342 (Molecular Probes, USA) to visualize the cell nucleus. Cells were imaged either under a 60× or 100× objective with a Nikon A1R inverted laser scanning confocal microscope (Nikon microsystems, France).

For the flow cytometry analysis, cells were incubated in a blocking solution (PBS containing 1% BSA) for 10 minutes. After centrifugation, cells were resuspended in the blocking solution and stained with the following monoclonal antibodies for 30 minutes: FITC anti-CD44, PE anti-CD24, PerCP anti-CD19, APC anti-CD45, and FITC anti-CD34. For intracellular staining, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 4% BSA. The cells were then stained with Oct-4 antibody (Cell Signaling Technology, USA), Sox2 antibody (R&D Systems, USA), Nanog antibody (Bioss Antibodies, USA), E-Cadherin antibody (Cell Signaling Technology, USA), N-Cadherin antibody (Abcam, USA), Snail + Slug antibody (Abcam, USA), ALDH1A1 antibody (Pierce Antibodies, USA), and β-Catenin antibody (Cell Signaling Technology, USA). Cells were then labeled with the appropriate Alexa Fluor® secondary antibodies (Molecular Probes, USA). Flow cytometry was carried out using FACSCalibur (Becton Dickinson, USA) following standard procedures with CellQuest Pro Software (Becton Dickinson, USA). Data analysis was performed using FlowJo v. 10.2 software (Treestar, USA) with super-enhanced Dmax (SED) subtraction analysis for determination of differences in histograms.

Nuclei of murine ESCs (24 x 10e6 cells/replicate) or differentiated neurons (day 10 after LiF withdrawal, 34 x 10e6 cells/replicate) from 2 biological replicates were extracted and cross-linked with 1.5% formaldehyde for 15 min, lysed and sonicated to solubilize and shear the crosslinked DNA. The resulting nuclear extracts were immuno-precipitated with the SOX2 antibody (R&D Systems, AF2018) overnight at 4°C. Eluted and de-crosslinked DNA was used to prepare sequencing libraries for Illumina using NEBNext Ultra II DNA Library Prep Kit for Illumina. Data were collected using 50 reads single-end mode on HiSeq2000.