Fluoview fv1000 confocal laser system

BLB integrity was assessed by evaluating the extravasation and diffusion of a non-permeable dye (Evan's blue; EBD) around strial capillaries. Under deep anesthesia with 10% chloral hydrate as aforementioned, 2% EBD (20 mg/ml/kg; E2129; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was injected into the femoral vein 2 h prior to sacrifice. The cochlea was perfused with 4% paraformaldehyde in 0.1 M PBS in the vicinity of the round and oval windows, and the stria vascularis gently dissected from the bony cochlear lateral wall and fixed overnight in 4% paraformaldehyde at 4°C. The degree of EBD extravasation in the stria vascularis was assessed by reading the fluorescence under an Olympus IX81 inverted microscope fitted with an Olympus Fluoview FV1000 confocal laser system. Image processing and fluorescence analysis of the images were performed using Image J 1.30 software.

Primary antibodies used in the experiment included monoclonal mouse anti-MMP-2 (catalog no. MAB3308; EMD Millipore, Billerica, MA, USA), polyclonal rabbit anti-MMP-9 (catalog no. AB19016; EMD Millipore) and polyclonal rabbit anti-ZO-1 (catalog no. 617300; Invitrogen; Thermo Fisher Scientific, Inc.). Secondary antibodies included Alexa Fluor 488-conjugated goat anti-mouse IgG (catalog no. A11001) and Alexa Fluor 568-conjugated donkey anti-rabbit IgG antibodies (catalog no. A10042), both purchased from Invitrogen; Thermo Fisher Scientific, Inc. Stria vascularis samples were fixed in 4% paraformaldehyde at 4°C for 2 h, washed in PBS for 30 min, permeabilized in 0.5% Triton X-100 for 1 h, and immunoblocked in a solution of 5% goat serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) in PBS for 1 h. The specimens were incubated overnight at 4°C with the primary antibody diluted (1:200) in PBS. After several washes in PBS, tissues were incubated with secondary antibodies (1:200) at room temperature for 1 h. The fluorescence was visualized under an Olympus IX81 inverted microscope fitted with an Olympus Fluoview FV1000 confocal laser system. The samples were examined as above, and Z-series stacks were acquired at 1-um intervals. The Z-series images were visualized using Image J 1.30 software (National Institutes of Health, Bethesda, MD, USA).