Rabbit anti sox2 antibody

For the detection of protein expression of DNMT1, Sox2, and β-catenin in human and animal tissues, 5 μm-thick paraffin or optimum cutting temperature (OCT) sections were stained by dual IF. The sections were incubated with primary antibodies (1:100 dilution of rabbit anti-DNMT1 antibody [Abcam, Cambridge, MA], 1:50 dilution of rabbit anti-Sox2 antibody [Santa Cruz Biotech, Santa Cruz, CA], 1:100 dilution of rabbit anti-β-catenin antibody [Santa Cruz Biotech, Santa Cruz, CA], 1:200 dilution of mouse anti-PTH antibody [Abcam, Cambridge, CA], and 1:500 dilution of pig anti-insulin antibody [Dako, Carpinteria. CA]) overnight at 4°C. Slides were then incubated with secondary antibodies (1:200 dilutions each of anti-mouse Alexa Fluor 647, anti-pig Alexa Fluor 647, and anti-rabbit Alexa Fluor 488; Invitrogen, Grand Island, NY, USA) for 45 minutes in the dark. The slides were then mounted in Vectashield mounting medium with 4′, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Images were taken using a fluorescence microscope with a camera.

Each animal was deeply anesthetized by intraperitoneal injection of pentobarbital (20 mg/kg), and then perfused with chilled phosphate-buffered saline, followed by 4% paraformaldehyde in 0.1 mol/l phosphate buffer. After post-fixation overnight, 50 µm thick sections were cut with a vibrating blade microtome (VT1000S; Leica, Wetzlar, Germany). For immunohistochemistry, the following primary antibodies were used: rabbit anti-GFP antibody (1:500, MBL, Nagoya, Japan), goat anti-GFP antibody (1:200, Abcam, Cambridge, UK), rabbit anti-Iba1 antibody (1:500, Wako, Osaka, Japan); rabbit anti-glial fibrillary acidic protein (GFAP) antibody (1:500, Dako, Glostrup, Denmark); goat anti-PDGFRα antibody (1:100, R&D Systems, MN, U.S.A); rabbit anti-GST-π antibody (1:500, Enzo life sciences, NY, U.S.A.); rabbit anti-nestin antibody (1:200, Santa Cruz Biotechnology, CA, U.S.A.); goat anti-doublecortin (Dcx) antibody (1:100, Santa Cruz Biotechnology); mouse anti-betaIII tubulin (Tuj1) antibody (1:100, Santa Cruz Biotechnology); mouse anti-NeuN antibody (1:100, Millipore, MA, U.S.A.); mouse anti-Ascl1 antibody (1:100, BD Pharmingen, NJ, U.S.A.); rabbit anti-Sox2 antibody (1:100, Santa Cruz Biotechnology); mouse anti-NeuroD1 antibody (1:100, Abcam). Each primary antibody was detected by appropriate secondary antibodies conjugated with Alexa Fluor 488 or 555^TM (Molecular Probes, OR, U.S.A.).