Avidin biotin horseradish peroxidase hrp

For immunohistochemical examination, paraffin-embedded colonic sections were deparaffinized in xylene and hydrated in gradient alcohol. Then, antigen retrieval was performed by heating in pre-boiling buffer in a microwave for 10 min. Next, slides were incubated in 3% hydrogen peroxide solution for 15 min to quench endogenous peroxidase activity and then blocked by 10% goat serum in PBS (pH 7.4) for 15 min at room temperature. Subsequently, slides were incubated with primary antibodies in a humidified chamber at 4°C overnight: cyclooxygenase-2 (COX-2; 1:300), inducible nitric oxide synthase (iNOS; 1:300), β-catenin (1:200), E-cadherin (1:200), N-cadherin (1:200; BOSTER, Wuhan, China), proliferating cell nuclear antigen (PCNA; 1:100), Vimentin (1:300), Snail (1:100; Bioss, Beijing, China), or p53 (1:50), p-p53 (1:50; Santa Cruz, Dallas, TX, USA). After incubation with biotinylated goat anti-rabbit secondary antibody (1:200; Beyotime, Jiangsu, China) and avidin-biotin-horseradish peroxidase (HRP; Beyotime, Jiangsu, China), slides were visualized using diaminobenzidine (DAB), counterstained with haematoxylin and observed under an optical microscope.

For immunohistochemistry, paraffin-embedded pulmonary sections were deparaffinized with xylene, hydrated in gradient ethanol, and microwaved for 10 min for antigen retrieval. After washing with PBS, the sections were incubated with 3% H₂O₂ for 15 min to quench the endogenous peroxidase activity and blocked with goat serum (Solarbio, Beijing, China) for 15 min at room temperature. Subsequently, the sections were incubated with primary antibodies against iNOS (1:200) and xyxlooxygenase-2 (COX-2; 1:200) (Boster, Wuhan, China) in a humidified chamber at 4°C overnight. After rinsing with PBS, the slides were incubated with biotinylated goat anti-rabbit secondary antibody (1:200; Beyotime) for 30 min at 37°C and then avidin-biotin- horseradish peroxidase (HRP; Beyotime) for 30 min at 37°C. Finally, the sections were visualized by adding diaminobenzidine (DAB), counterstained with hematoxylin, and examined under light microscope.