The sections were permeabilized with a 0.1% Tween 20 (Sigma-Aldrich, Milan, Italy) solution in PBS, endogenous peroxidase was blocked with 1% hydrogen peroxide (Marco Viti Pharmaceutical, Sandrigo, Vicenza, Italy), and non-specific sites were saturated with the blocking buffer Vecstain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). Sections were then incubated with the primary antibody at the concentrations 1:250 for IL-6 (bs-0782R, Bioss Inc, Woburn, Massachusetts, USA) and 1:200 for TNFalfa (ab1793, abcam, San Francisco, USA) overnight at 4°C.
After 24 h, the incubation with the appropriate secondary antibody Vecstain Elite ABC kit (Vector Laboratories) was performed, followed by signal amplification and detection with Vector SG Peroxidase substrate kit (Vector) and the Nuclear Fast Red (Vector Laboratories). Finally, after dehydration and a passage in xylene, the slides were mounted with Surgipath® Micromount (Leica Biosystem, Buccinasco, Milan, Italy) and observed with the optical microscope ECLIPSE 50i (Nikon). Two samples per groups were analyzed at the cochleostomy and mid-modiolar region (about 15 sections each). The images were acquired with Nis Elements D 3.2 software (Nikon).
After 24 h, the incubation with the appropriate secondary antibody Vecstain Elite ABC kit (Vector Laboratories) was performed, followed by signal amplification and detection with Vector SG Peroxidase substrate kit (Vector) and the Nuclear Fast Red (Vector Laboratories). Finally, after dehydration and a passage in xylene, the slides were mounted with Surgipath® Micromount (Leica Biosystem, Buccinasco, Milan, Italy) and observed with the optical microscope ECLIPSE 50i (Nikon). Two samples per groups were analyzed at the cochleostomy and mid-modiolar region (about 15 sections each). The images were acquired with Nis Elements D 3.2 software (Nikon).