Immunohistochemistry of Human Cochlear Tissues

The tissue preparation for paraffin embedding, the immunohistochemistry procedure and the digital examination of human cochlear sections were described in detail in our previous publications [61 (link),62 (link),63 (link),64 (link),65 (link)]. Negative controls were acquired by substituting the primary antibodies with isotype-matching immunoglobulins. These negative controls did not yield any immunostaining. Immunohistochemistry was performed utilizing a Ventana Roche XT immunostainer (Mannheim, Germany), applying a DAP-MAP discovery research standard procedure. Then, 5 µm thick human inner ear FFPE sections were incubated for 1 h at 37 °C with primary antibodies, followed by 1 h at 37 °C with Universal Secondary Antibody (supplied from Ventana, Roche, Mannheim, Germany, 760-4250). The primary antibodies were OTOF (polyclonal, rabbit, dilution 1:200, Invitrogen, Karlsruhe, Germany, PA5-79776) and TECTA (polyclonal, rabbit, dilution 1:150, Invitrogen, PA5-80102).