Rabbit anti nanog

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).

Most of the reagents used in cell culture were obtained from Gibco (UK) unless specifically indicated. The primary and secondary antibodies were as follows: Rat anti-SSEA-3 (Invitrogen, USA), phycoerythrin (PE)-SSEA-4 (eBioscience, USA), mouse anti-TRA1-60 (Millipore, USA), PE-OCT-4 (BD Pharmingen, USA), rabbit anti-Nanog (Abcam, USA), fluorescein isothiocyanate (FITC)-goat anti-rabbit Ig (Abcam,), FITC-goat anti-mouse (Biorad, USA), FITC-conjugated sheep anti-mouse Ig (Sina biotech, Iran), and HRP-conjugated sheep anti-mouse Ig (Sina biotech). Extracellular vesicles-specific antibodies were from SBI system biosciences, CA, USA. Extracellular vesicle-depleted fetal bovine serum was purchased from Gibco. PKH-26 dye labeling kit was from Sigma (USA). Annexin V/PI apoptosis kit, ECL, BCA protein assay kit and calcein-acetoxymethyl (cAM) were purchased from ebioscience (USA), GE healthcare (USA) and BD Pharmingen, respectively.

ChR2-GFP-V6.5 ESCs seeded on Menzel-glass coverslips were fixed by 4% paraformaldehyde for 30 min and washed three times with PBS (5 min per wash). Then, the cells were permeabilized with 0.3% Triton and blocked with 10% bovine serum albumin (BSA) at 37 °C for 60 min and then covered with primary antibody solution and transferred to a cold room at 4 °C overnight. The cells were washed three times, and secondary goat anti-mouse antibody conjugated to Alexa Fluor 555 (Molecular Probe, USA) was added for incubation at 37 °C for 60 min. After the cells were washed three times with PBS, 4′,6-diamidino-2-phenylindole (DAPI) was added for 10 min, and the cells were washed in water three times and mounted using Fluoromount (Dako, Denmark). The primary antibodies were mouse anti-OCT4 (Chemicon, 1:200), Rabbit-anti SOX2 (Abcam, 1:200), Rabbit-anti NANOG (Abcam, 1:200), Mouse-anti Ki67 (Abcam, 1:200) and mouse anti-SSEA1 (Chemicon, 1:200). The images were acquired with a Leica TCS SP2 inverted confocal microscopy system with an HCX PL APO CS 40× 1.25 NA oil immersion objective (Leica, USA). Immunostaining semi-quantification between non-light and light stimulation groups was done as previous [19 (link)].

The expression of pluripotent marker proteins in MSCs derived from dental pulp, papilla, and follicle tissues was evaluated by Western blotting. In brief, the lysates of protein were prepared from cultured MSCs using RIPA buffer (Thermo Scientific, Rockford, IL, U.S.A.) containing protease inhibitors. The concentration of protein was measured using Microplate BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL, U.S.A.) and a total of 25 μg protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Mini Protean, BioRad, Hercules, CA, U.S.A.). The separated proteins were then transferred onto polyvinylidene difluoride membranes (PVDF, Millipore) and membranes were incubated with primary antibodies such as rabbit anti-OCT4 (1:200, abcam), rabbit anti-SOX2 (1:200, abcam), rabbit anti-NANOG (1:200, abcam), and mouse anti-GAPDH (1:200, Millipore) for overnight at 4°C. Then membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000, Santa Cruz Biotechnology), and goat anti-mouse IgG (1:10,000, Santa Cruz Biotechnology) secondary antibodies for 2 h at room temperature. The immunoreactivity was detected in dark condition by adding enhanced chemiluminescence (ECL; Supersignal, West Pico Chemiluminescent substrate, PIERCE, IL) and exposed to X-ray films.