Immunohistochemical Analysis of Tissue Samples

Immunohistochemistry was performed as described previously (Tu et al. 2017 (link)). Tissues samples of the cortex and myocardium were fixed with 4% polyformaldehyde, embedded in paraffin, cut into slices (thickness, 10 μm), dewaxed and hydrated. Then the slices were incubated with 3% hydrogen peroxide, washed with PBS, blocked with 10% normal goat serum at 37 °C for 30 min, incubated with rabbit polyclonal antibody for CAIII (1:200; Abcam), P53 (1:100; Abcam), IL-17 (1:1000; Abcam), in PBS containing 3% BSA overnight at 4 °C, followed by incubation with biotinylated secondary anti-rabbit antibody at 37°Cfor 45 min. Immunohistochemical staining followed by diaminobenzidine staining was subsequently performed. Finally, all sections were dehydrated sequentially with 75% ethanol for 5 min, 85% ethanol for 5 min, ethyl alcohol twice for every 5 min, and xylene for 6 min. They were then covered on slides for image analysis.