Rabbit anti sox2

The cochleae of Sprague-Dawley rats at P1 were dissected from the temporal bone as described above, and then were fixed with 4% paraformaldehyde solution for 30 min at room temperature. Then the basilar membranes were isolated and thoroughly rinsed with 0.01 M PBS. We modified the protocol of immunofluorescence as described in our previous studies.^{12 (link),13 (link)} They were in a blocking/permeabilization solution [10% normal donkey serum (Jackson Labs, Bar Harbor, ME, USA) and 0.3% Triton X-100 in 0.01M PBS] for 30 min at room temperature. They were subsequently incubated with goat anti-myosin VIIa (1:300, Proteus BD BioSciences, San Jose, CA, USA) and rabbit anti-Sox2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies in 5% normal donkey serum and 0.1% Triton X-100 in 0.01 M PBS overnight at 4°C, mediated by DyLight 594 and DyLight 488 conjugated secondary antibodies (goat anti-rabbit IgG or rabbit anti-goat IgG) at a concentration of 1:500 for 1 h at room temperature. Observation results were recorded under a fluorescence microscope after sealing slices. Images were photographed on a laser confocal scanning microscope (LSM 710, Zeiss, Jena, Germany) with a 20× lens by using Zeiss ZEN 2010 software. Images were processed using Adobe Photoshop software.

Protein lysate of all the samples was prepared using RIPA buffer (Thermoscientific) containing protease inhibitors. Protein concentration was determined by Microplate BCA Protein Assay kit (Thermo Scientific), a total of 25 μg each protein sample was separated using 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, Mini Protean, BioRad) and transferred onto polyvinylidene difluoride membranes (PVDF, Millipore, USA). Membranes were then incubated with primary antibodies of goat anti-Oct-3/4 (43–50 kDa, 1:200, Santa Cruz), rabbit anti-Sox-2 (34 kDa, 1:200, Santa Cruz), goat anti-Nanog (35 kDa, 1:200, Santa Cruz), rabbit anti-Bax (22 kDa, 1:1000, Enzo, NY, USA), rabbit anti-Bcl-2 (28 kDa, 1:1000, Cell Signalling, MA, USA), mouse anti-p53 (53 kDa, 1:200, Santa Cruz), rabbit anti-p21 (21 kDa, 1:200, Santa Cruz) and rabbit anti-β actin (45 kDa, 1:1000, Cell Signalling) for overnight at 4°C followed by incubation with horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG (1:10000, Santa Cruz), goat anti-rabbit IgG (1:10000, Santa Cruz) and goat anti-mouse IgG (1:10000, Santa Cruz) secondary antibodies for 1 h at room temperature. Immunoreactivity was detected by enhanced chemiluminescence (ECL; Supersignal, West Pico Chemiluminescent substrate, PIERCE, IL, USA) and exposed to x-ray films.

Tissues were obtained from three to five mice and prepared for paraffin sections (5 μm). For immunofluorescent staining, tissues were dehydrated and then stained as detailed previously 2 using rabbit anti‐SOX2 1:400, mouse anti‐P63 1:100 (Santa Cruz Biotechnology, USA), goat anti‐K12 1:400 (Santa Cruz Biotechnology, USA), rabbit anti‐K15 1:500 (Abcam, UK; overnight at 4°C). Next, samples were washed (0.2% tween 20, 0.2% gelatine), incubated with secondary antibody (Alexa Fluor 488 and 594 [Invitrogen, USA]), and mounted as above. In situ hybridization for miR‐450b was performed on whole embryos or on optimal cutting temperature (OCT) compound frozen sections as described previously 25.