Masson’s trichrome staining and the morphometry

The liver samples were isolated from the liver fibrosis mice sacrificed on day 0, 30 and 45 upon injection with CCl₄ and then fixed in 4% paraformaldehyde and embedded in paraffin, followed by frozen section. For the Masson’s trichrome staining of the liver sections, Trichrome Stain Kit (Abcam, Catalog#ab150686) was used. Briefly, the sections were firstly deparaffinized and rehydrated in distilled water. Then, the sections were incubated successively with preheated Bouin's Fluid, Weigert's Iron Hematoxylin, Biebrich Scarlet/Acid Fuchsin solution, phosphomolybdic/phosphotungstic acid solution, Aniline Blue solution and acetic acid solution, with rinse steps at the interval of every two incubation steps. The duration of each incubation step was controlled according to the recommendatory procedures of the manual. The stained sections were then carefully observed and photographed under microscope (Olympus). The proportion of each staining area was analyzed by ImageJ software. The blue staining area represents the collagen-enriched tissues, which was considered as the fibrosis area of the livers.