assay
The wet weight of the liver or spleen was measured and then divided by the body weight to obtain the liver or spleen index. Then, the liver tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections (4-μm thick) were prepared and placed on glass slides. They were then stained with hematoxylin and eosin (H&E) or Masson’s trichrome stain following standard protocols. A pathologist who was blinded to the experimental groups scored liver fibrosis under a microscope according to the METAVIR scoring system (F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis with rare septa; F3, numerous septa without cirrhosis; F4, liver cirrhosis) (16).
The expression of α-SMA in the liver was detected by immunohistochemistry
Tissue sections (4-μm thick) were prepared and placed on glass slides. Endogenous enzymes were inactivated, and antigen retrieval was performed using heat (in 0.01 M citrate salt buffer, pH 6.0, heating to 100°C two times for 10 mins). Then, the sections were incubated with 5% BSA at room temperature for 20 min, followed by the addition of anti-α-SMA (1:40) antibody (Santa Cruz Biotechnology, Inc., CA, USA) at 2–8°C overnight. They were then incubated with biotinylated goat anti-rabbit IgG (1:100) (Wuhan Boster, Wuhan, Hubei, China) at 37°C for 30 min. Next, streptavidin-biotin complex (SABC) detection reagent (1:100) (Wuhan Boster, Wuhan, Hubei, China) was added dropwise, and the samples were incubated for 30 min before 3,3’-diaminobenzidine (DAB) reagent (Wuhan Boster, Wuhan, Hubei, China) was added. After washing with distilled water, the sections were stained with hematoxylin, and a pathologist who was blinded to the treatment groups counted the number of positively stained cells under a microscope.