Fluorescent mounting medium

Decalcified cochleae were rinsed three times (30 minutes per rinse) in phosphate buffer saline (PBS). The cochleae were then immersed in 30% sucrose overnight for cryoprotection and embedded in OCT compound for cryo-sectioning at −20 °C. Cryosections with a thickness of 10 µm were washed three times before blocking for one hour at room temperature in blocking solution containing 10% goat serum in PBST (0.1% Triton X-100 in PBS). They were then incubated overnight at 4 °C in mouse anti-Tuj1 (Covance) and rabbit anti-peripherin (Millipore), both in an antibody solution of PBS +3% goat serum +0.1% Triton X-100. Sections were washed three times with PBST and then incubated for 2 hours at room temperature in Alexa Fluor 647 anti-mouse, Cy3 goat anti-rabbit, and FITC goat anti-rabbit (Jackson ImmunoResearch Laboratories, INC.). Specimens were washed three times, 5 minutes each, in PBS before mounting with fluorescent mounting medium (Electron Microscopy Sciences).

After fixation, neurosphere-derived differentiated cells were washed three times in PBS and permeabilized in PBS containing 0.48% (vol/vol) Triton X-100 (Sigma) for 5 min at RT. The primary antibodies against MAP-2 (rabbit polyclonal, 1 : 600, Chemicon, Temecula, CA), GFAP (mouse monoclonal, 1 : 600, Millipore, Billerica, MA), or NG2 (rabbit polyclonal, 1 : 500, Abcam, Cambridge, MA) were incubated for 150 min at RT in an antibody solution containing 16% (vol/vol) goat serum. Secondary antibodies were as follows: Alexa Fluor 488 conjugated goat anti-rabbit antibody(1 : 1600, Invitrogen) and Alexa Fluor 488 conjugated goat anti-mouse antibody (1 : 1600, Invitrogen). Nuclei were counterstained with DAPI (1 : 10000, Sigma). Fluorescent Mounting Medium (Electron Microscopy Sciences, Hatfield, PA) was applied to slides as antifading agent prior to addition of coverslips.