Immunohistochemical Procedures for Protein Analysis

Immunohistochemical procedures were conducted according to our previous study [52 (link)]. Briefly, the deparaffinized and hydrated slides were incubated in 3 % H₂O₂ for 30 min to block the endogenous peroxidase activity and were later placed in citrate buffer (PH = 6) for antigen retrieval. The specimens were incubated with the primary antibody overnight at 4°C (p-IκBα, 1: 100; LC3B, 1: 100; Beclin-1, 1: 100, Thermo Fisher Scientific, USA; p-NF-κB p65, 1: 100; caspase-9, 1: 50; p-Akt, 1: 100; and TLR4, 1: 100; Abcam, U.K). After three washes with 0.1 mol/L PBS, the sections were incubated with a biotinylated goat anti-rabbit immunoglobulin G secondary antibody (Zhongshan Goldenbridge Biological Technology, China) at 30°C for 25 min. Next, the specimens were incubated with a streptavidin-biotin complex at 30°C for 20 min and were further incubated with diaminobenzidine for 15 min at room temperature. The slides were counterstained with hematoxylin and were visualized under a microscope at a magnification of 400× (Olympus, Germany). The positive cell counts were analyzed in five independent sections and were captured using a pathological image analyzer (Leica DM6000, Germany). The immunopositive cell rate was calculated as the number of immunopositive cells divided by the total cells (including immunopositive cells and immunonegative cells).