2.7. Immunohistochemical Analysis

Sections of formalin-fixed and paraffin-embedded tissues were analyzed via immunohistochemistry (IHC). In brief, slices were incubated with 3% hydrogen peroxide solution to block endogenous peroxidase activity; treated with boiling citrate buffer, pH 6.0 (Wuxi Aorui Dongyuan Biotechnology Co., Ltd., Wuxi, China; ZLI-9065), for 6 minutes for antigen repair; and blocked with 10% goat serum (Solarbio Co., Dalian, China, SL038) for 20 minutes. Next, sections were incubated with anti-AAT (Proteintech Group Inc., Wuhan, China; 66135-1-lg, 1 : 300), anti-RAB2B (Proteintech Group Inc., 11756-1-ap, 1 : 250), and anti-IGFBP2 (Beijing BIOSS Biotechnology Co., Ltd., Beijing, China; bs-1108r, 1 : 100) antibodies at 37°C for 1 hour, followed by enhanced anti-rabbit biotin IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China; PV-9000) for 20 minutes, and finally DAB solution (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; ZLI-9017) for color development for AAT (90 seconds), RAB2B (120 seconds), and IGFBP2 (120 seconds). Stained sections were identified and scored by two high-level pathologists as follows: staining area score < 26% (1 point), 26-50% (2 points), 51-75% (3 points), or 76-100% (4 points) and staining intensity score of light yellow (1 point), brownish yellow (2 points), or brown (3 points). The final score was calculated as the staining area score multiplied by staining intensity score [7].