Immunohistochemical Analysis of ER Stress Markers

Liver tissues were fixed in 4% paraformaldehyde, dehydrated, and subjected to heat-induced antigen retrieval using citrate. Then, the sections were incubated in 1% Triton X-100 for 15 min. After eliminating endogenous peroxidase activity with 3% hydrogen peroxide for 15 min, the sections were blocked with goat serum albumin at room temperature for 1 h. Then, the sections were incubated overnight at 4°C with p-IRE1α (cat. no. ab48187; 1 : 100; Abcam Inc.), TRAF2 (cat. no. #4712; 1 : 100; CST Inc.), and p-p65 (cat. no. #3033; 1 : 100; CST Inc.). The sections were washed and incubated with species-matched secondary antibodies (1 : 200 dilution) for 1 h at room temperature. The sections were washed with PBS, treated with 3,3′-diaminobenzidine for 5 min at room temperature, stained with hematoxylin for 30 sec at room temperature, and washed with flowing water for seconds. Following dehydration, sections were sealed with neutral resin, and specific staining was visualized by light microscopy as described before [22 (link)].