Mouse anti myelin basic protein

The temporal bones were removed and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1.5 hr at 4°C. The temporal bones were decalcified by incubation in 10% EDTA at 4°C for 3–5 days. The EDTA solution was changed daily. The bones were then embedded in the OCT compound for cryostat sectioning. The sections of 10 μm thickness were washed in PBS, and nonspecific binding was blocked with 1% bovine serum albumin (BSA) and 10% goat serum in PBS plus 0.1% Triton X-100 (PBST) for 1 hr. The primary antibodies, chicken anti-Tuj1 (Abcam), mouse anti-myelin basic protein (Abcam), rabbit anti-Myo7a (Proteus Biosciences, Inc), mouse anti-Tuj1 (Abcam), rabbit anti-TRPA1 (Abcam), TRPC3 (Novus Biologicals), TRPC6 (Abcam), TRPV1 (Novus Biologicals), TRPV4 (Abcam), were incubated overnight at 4°C. After incubating the primary antibodies, the slides were washed three times with PBST and incubated with secondary antibodies for 1.5 hr at RT in the dark. We used Alexa Fluor 647-conjugated goat anti-mouse and Cy3-conjugated goat anti-chicken, Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 568-conjugated goat anti-mouse (Jackson ImmunoResearch Labs) in a dilution of 1:500. Other markers used were phalloidin-Fluor 647 (Abcam) for F-actin and DAPI (Sigma) for nuclear stain. The slides were then examined under a confocal microscope (LSM 510, Zeiss).

After deparaffinization and heat-mediated antigen retrieval (EDTA-based pH 9.0 solution), as previously described, the tissue sections were blocked with goat serum for 30 min at 37°C and then incubated at 4°C overnight with primary antibody (mouse anti-myelin basic protein (MBP) monoclonal antibody, 1 : 400, Abcam, USA; rabbit anti-synaptophysin monoclonal antibody, 1 : 100, Abcam, USA; goat anti-Olig2 polyclonal antibody, 1 : 50, R&D, USA). Sections were washed three times with 0.1 M phosphate buffered saline (PBS) for 5 min before incubation with secondary antibodies (donkey anti-rabbit IgG, 1 : 500, Abcam, USA; donkey anti-mouse IgG, 1 : 500, Abcam, USA; donkey anti-goat IgG, 1 : 200, Abcam, USA) for 4 h at room temperature and 4′,6-diamidino-2-phenylindole (DAPI; 1 : 500, Sigma-Aldrich) sequentially for nuclear staining. Immunofluorescent images were visualized and photographed using a confocal laser-scanning microscope (C1; Nikon, Tokyo, Japan).