Purified anti-Tubulin β-3 (TUBB3) Antibody (Previously Covance catalog# PRB-435P)

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Publication protocol

The immunohistochemical procedures were applied to intact utricles. Specimens were first immersed in blocking solution for 2 h at room temperature containing 0.1 % Triton X-100 and 1.0 % BSA solution in PBS, and were then incubated for 24 h at 4 °C in cocktails of the primary antibodies diluted in blocking solution. Fluorophore-conjugated, host-specific secondary antibodies were then applied for 2 h at room temperature. Utricles labeled with anti-OCM, anti-TUBB3, and anti-Calb2 were used to investigate the relationship between OCM-positive (OCM+) hair cells, TUBB3-positive (TUBB3+) calyces, and CALB2-positive (CALB2+) calyces. Fluorophore-conjugated phalloidin (Biotium CF405M) was added to the secondary antibody solutions for stereocilia labeling, which provided the basis for determining hair cell morphologic polarization and navigating the topography of the utricle during confocal imaging. Secondary antibodies were used at 1:250 concentration and included Alexa 488 donkey anti-mouse (Life Technologies A21202), Alexa 555 donkey anti-goat (Life Technologies A21432), and Alexa 647 donkey anti-rabbit (Life Technologies A31573). After the final washes in PBS, intact utricles were whole-mounted on glass slides with aqueous mounting media. Silicone spacers (S-24735; Invitrogen) were used to create a small chamber on the glass slide within which the utricle specimens were mounted, thereby minimizing distortion of in situ structure by the placement of the coverslip.

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