Mouse anti tuj1

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).

The purified SSCs and different PCL membranes were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.25% Triton X-100 for 10 min. Then samples were blocked with 10% blocking buffer for 1 h and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-UCHL-1 (Abcam, 1:200), mouse anti-GFRα-1 (Abcam, 1:200), mouse anti-Tuj-1 (Abcam, 1:200). After washing with PBS, the samples were incubated in Alexa Fluor 488 or Alexa Fluor 594-conjugated secondary antibodies at 37 °C for 2 h, and DAPI was used to stain the nuclei. Finally, the samples were observed under a laser confocal scanning microscope.