Alexa fluor 555 goat anti mouse igg1

Animals were transcardially perfused with 4% paraformaldehyde in 0.1 M PBS following flushing with prewarmed PBS. The cochleae of the guinea pigs were removed and immersed in 4% paraformaldehyde for 1 h at RT. The organ of Corti was carefully dissected and permeabilized with 5% horse serum in PBS supplemented with 0.3% Triton X-100 for 1 h. The samples were then incubated with anti-CtBP2 IgG1 (1:100; 612044, BD Biosciences, San Jose, CA, USA), anti-GluA2 IgG (1:1000; MAB397, Merck KGa, Darmstadt, Germany) and anti-myosin 7a (1:100; sc-74516, Santa Cruz Biotechnology Inc., Dallas, TX, USA) polyclonal antibodies with 3% horse serum in PBS supplemented with 0.3% Triton X-100 overnight at 37 °C. After three washes with PBS, the tissues were incubated with secondary antibodies against CtBP2 (Alexa Fluor™ 555 goat anti-mouse IgG1, 1:500, A21127, Thermo Fisher Scientific, Waltham, MA, USA), GluA2 (Alexa Fluor™ 488 goat anti-mouse IgG2a, 1:1000, A21131, Thermo Fisher Scientific, Waltham, MA, USA), and myosin 7a (Alexa Fluor™ 647 donkey anti-rabbit IgG (H+L), 1:500, A31573, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37 °C. The samples were mounted with DAPI Fluoromount-G^® mounting medium (SouthernBiotech, Birmingham, AL, USA) and covered with a coverslip for analysis. Images were obtained using an LSM 880 Zeiss confocal microscope.

The following primary antibodies were used in this study: human collagen type I (Col1) rabbit immunoglobulin poly IgG (1:200) (ACRIS, SAN, CA, USA), collagen type II (Col2) clone 6B3 (1:200) (Merck, Darmstadt, HE, Germany). The following second antibodies were used: Alexa Fluor 488 goat anti-rabbit IgG1 (1:500) (Invitrogen: Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 555 goat anti-mouse IgG1 (1:500) (Invitrogen). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).