Alexa 488

In both ND and ED noise-exposed and ND-CTR and ED-CTR unexposed rats, the right cochleae were cryoprotected in 30% sucrose in PBS, frozen at −70 °C by immersion in a 2-propanol/dry ice bath, and further encased in a 15% sucrose and 10% gelatin solution. Blocks were sectioned parallel to the modiolus in a cryostat at a thickness of 20 mm. Sections in the modiolar plane were mounted serially on gelatin-coated slides and processed for immunohistochemistry. After several rinses in PBS containing 0.2% Triton X-100 (Tx), sections were incubated for 1 h in PBS-Tx (0.2%) with 10% normal goat serum (NGS). Next, sections were incubated overnight in a humid chamber at 4 °C with the corresponding primary antibodies (anti-CAT, GPX1, SOD1, and BCL-2; see Table 2) diluted in a solution containing PBS-Tx (0.2%), pH 7.4. The next day, after four 15 min rinses in PBS-Tx (0.2%), sections were incubated for 2 h in the corresponding fluorescent secondary antibody conjugated to Alexa 488 (1:200, Vector Laboratories, Burlingame, CA, USA) and also in biotinylated phalloidin (Pha) which was then visualized with streptavidin conjugated to Alexa 594 (Molecular Probes, Eugene, OR, USA). Finally, sections were counterstained with DAPI nuclear staining and cover slipped. Immunofluorescence was visualized using a laser scanning confocal microscope as outlined in the previous section.