Mab1527

For cochlear whole-mounts and neuronal cultures, preparations were fixed and permeabilized with 100% methanol at −20°C for 6 min and rinsed three times with 0.01M PBS (pH 7.4). Before the application of antibodies, specimens were incubated with 5% normal goat serum (NGS) for 1h at RT or 4°C overnight. Specimens were incubated with primary antibodies at either 4°C overnight (peripherin, calretinin and calbindin) or at RT for 1h (β-tubulin). Monoclonal mouse anti-β-tubulin (1:200, Covance, MMS 435P) was used to label all neurons and monoclonal mouse anti-peripherin (Chemicon, MAB1527) antibody to specifically identify putative type II spiral ganglion neurons. Polyclonal rabbit anti-calretinin (Millipore, AB5054) was used at 1:200. Polyclonal rabbit anti-calbindin (Swant, CB38) and monoclonal mouse anti-calretinin (Millipore) antibodies were used at 1:100. After washing 3X with PBS the preparations were incubated with 5% NGS for 1h at RT and then with one of the following secondary antibodies at 1:100 for 1h at RT: Alexa Fluor 594 conjugated goat anti-mouse; Alexa Fluor 488 conjugated goat anti-rabbit, Alexa Fluor 350 conjugated goat anti-mouse (Invitrogen). Multi-antibody-labeling was done sequentially. At the end of all the staining steps the preparations were washed with 0.01M PBS and mounted in DABCO medium (Sigma-Aldrich).

Monoclonal mouse anti-peripherin antibody (Chemicon, MAB1527) was used as a marker for type II spiral ganglion neurons according to previous reports (Mou et al., 1998 (link); Reid et al., 2004 (link)). The appropriate concentration of anti-peripherin antibody was determined using a preparation in which the organ of Corti and the ganglion were cultured for 3 days in vitro, such that the type I and type II afferent innervations patterns were retained and could be distinguished using anti-peripherin antibody staining (Flores-Otero and Davis, 2011 (link)). Because anti-peripherin antibody also labels type I neurons in the apex when concentrations are too high or fails to label all type II neurons in the base when the concentrations are too low, we utilized the organ of Corti cultures to establish working concentrations that were optimized for different tonotopic regions (1:6000 for apex, 1:5000 for middle and 1:4000 for base).