Universal secondary antibody

Skin tissue sections (3 μm thick) were mounted onto silane-coated slides. The sections were deparaffinized by passing through xylene and a graded alcohol series before being stained with hematoxylin and eosin. The number of hair shafts per follicular unit was counted using a fluorescence microscope (FSX100; Olympus, Tokyo, Japan). In immunohistochemistry, tissue sections were heated in a microwave oven to 95 °C in citric acid buffer (pH 6.0) for 15 min for antigen retrieval, and then incubated with anti-Ki67 antibodies (cat. no. ab15580; Abcam, Cambridge, MA, USA; 1:100 dilution, v/v) at 4 °C overnight. The tissue sections were then incubated at room temperature for 60 min with a universal secondary antibody (Roche, Basel, Switzerland). The site for peroxidase binding was determined using DISCOVERY DAB Map Detection Kit (Roche). Sections were then counterstained with Hematoxylin II (Roche) for microscopic examination. As a negative control, nonimmune γ-globulin was used instead of the antibody. Images were captured using a fluorescence microscope (FSX100). Ki67-positive cells were counted, and the percentage was calculated relative to nuclear staining.

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).

The primary antibody, CD34 (dilution of 1:2500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), was used with a universal secondary antibody (dilution of 1:300) (Roche, Basel, Swizerland). The MVD index was calculated by taking five random pictures of each slide at a magnification of 200 × (10 ocular × 20 objective). The index was calculated as the number of CD34-stained cells divided by the total number of cells.