Immunohistochemistry, confocal, and 2-color stimulated emission depletion (STED) microscopy

Freshly dissected apical turns of the organs of Corti of 3 week old RIM-BP2^+/+ and RIM-BP2^−/− mice were either fixed with 4% formaldehyde (FA) in phosphate buffered saline (PBS) on ice for 10 min or with methanol at −20°C for 2 min. Thereafter, the tissue was washed 3 × 10 min in PBS and incubated for 1 h in goat serum dilution buffer (GSDB: 16% normal goat serum, 450 mM NaCl, 0.3% Triton X-100, 20 mM phosphate buffer, pH 7.4) in a wet chamber at room temperature. Primary antibodies were dissolved in GSDB and applied overnight at 4°C in a wet chamber. After washing 3 × 10 min (wash buffer: 450 mM NaCl, 20 mM phosphate buffer, 0.3% Triton X-100) the tissue was incubated with secondary antibodies in GSDB in a wet light-protected chamber for 1 h at room temperature. Then, the samples were washed 3 × 10 min in wash buffer and 1 × 10 min in 5 mM phosphate buffer, placed onto the glass microscopy slides with a drop of fluorescence mounting medium (Mowiol) and covered with thin glass coverslips. The following antibodies were used: mouse-IgG1-anti-CtBP2 (also recognizing the ribbon protein RIBEYE, BD Biosciences, 1:200), guinea pig-anti-Synapsin1/2 (Synaptic Systems, 1:500), rabbit-anti-Ca_V1.3 (Alomone Labs, 1:50), guinea pig-anti-bassoon (Synaptic Systems, 1:500), rabbit-anti-GluA2/3 (Chemicon, 1:200), rabbit-anti-RIM-BP2 (Synaptic Systems, 1:200) as well as secondary AlexaFluor568- and AlexaFluor647-labeled goat-anti-mouse and goat-anti-rabbit antibodies (Invitrogen, 1:200) and STAR580-, and Star635P-labeled goat-anti-mouse, goat-anti-rabbit and goat-anti-guinea pig antibodies (Abberior, 1:50). Confocal images were acquired using a laser scanning confocal microscope (Leica TCS SP5, Leica Microsystems GmbH, Mannheim, Germany) equipped with 561 and 633 nm lasers for excitation and a 63x oil immersion objective (1.4 NA, Leica). STED images were acquired using a 2-color STED microscope (Abberior Instruments, Göttingen, Germany) equipped with 561 and 640 nm excitation lasers, a 775 nm laser for STED (1.2 W) and a 100x oil immersion objective (1.4 NA, Olympus). Samples were treated in parallel and images were acquired in parallel, using same laser power, gain, and microscope settings. Images were analyzed using ImageJ, Igor Pro, OriginPro and Imaris software, and assembled for display in Adobe Illustrator software.