Immunohistochemistry

Liver tissue of each mouse which was fixed in 10% neutral buffered formalin solution was paraffin-embedded before routine histological staining. The prepared slides were dewaxed with xylene, dehydrated with alcohol, and then microwaved with sodium citrate buffer for 15 min to obtain the antigen. 3% hydrogen peroxide was incubated for 10 min to eliminate endogenous peroxidase activity after antigen repaired. These sections were then incubated overnight with a primary antibody against Bmal1 (1:200, ab272705, Abcam, United Kingdom) at 4°C. After washing with PBS, the sections were incubated with HRP-labeled broad-spectrum secondary antibody at room temperature for 20–30 min. The expression of Bmal1 was observed by 3,3′-diaminobenzidine tetrahydrochloride (DAB) staining. The sections were dehydrated after re-stained with hematoxylin for 3 min and then observed the positive region distribution of Bmal1 in the site of alcoholic liver injury.