Quantitative Immunohistochemistry Analysis of Vestibular Tissues

A DAB Detection Kit (Streptavidin-Biotin) kit was applied to examine the expression of the OCT-embedded vestibular apparatus tissue. Briefly, the endogenous peroxidase activity was blocked by incubation for 30 min in 0.3% H₂O₂. Nonspecific binding was blocked by a 20 min incubation with 10% normal goat serum. The sections were stained with different primary antibodies as listed in Supplementary Table 3 overnight at 4 °C. After rinsing with PBS and incubation with biotinylated anti-rabbit or anti-mouse IgG for 30 min at a dilution to 5 μg/ml, the sections were rinsed again with PBS and incubated with DAB complex for 1 min. The sections were counter-stained with hematoxylin, dehydrated, and omitted with Neutral balsam. Images were obtained using a Leica microscope at 40 × 10 magnification.
To evaluate the intensity of immunohistochemical reaction quantitatively, digital images were obtained and analyzed using a public domain ImageJ software. Intensity measurements were represented as the number in a 256-gray scale. Optical density values were corrected by subtracting the average values of background noise (mean background intensity) obtained from five image inputs. The optical density was then standardized by setting the threshold (mean background intensity) levels. Manipulation of the images was restricted to threshold and brightness adjustments to the whole image. At least 3 separate measurements per group were subjected to image analysis.