Immunofluorescence analysis of cochlear vasculature

The cochlear lateral wall tissues were collected and fixed in 4% paraformaldehyde in PBS for 1 h at room temperature. Tissues were permeated and blocked with 0.3% Triton X-100/10% normal goat serum/PBS for 1 h and then incubated with PECAM1 (Millipore, Burlington, MA, USA), MAB 13982, IgG) antibody at a concentration of 1:200 in blocking solution overnight at 4 °C. After rinsing six times in PBS for 10 min, Alexa Fluor 594 goat anti rat IgG (A11007, 1:200, Thermo Fisher Scientific, Waltham, MA USA) was used as a secondary antibody for PECAM. The specimens were mounted on glass slides using 50% glycerol and observed using a confocal microscopy TCS SP8 (Leica Microsystems, Wetzlar, Germany). The thickness of the stria vascularis was measured, as described previously [104 (link)], using image analysis software (Case Viewer, version 2.1, 3DHISTECH, Ltd. Budapest, Hungary).