Masson s trichrome staining

Colon tissues from mice with DSS‐induced colitis or colonic tumors were fixed in 4% paraformaldehyde and embedded in paraffin blocks. The paraffin blocks were sectioned (5 µm) for HE staining (Beyotime Biotech) or Masson's trichrome staining (Leagene) followed by coverslipped. Images were acquired using an Aperio VERSA 8 (Leica) multifunctional scanner. The pathogenic scores of the inflamed colons were evaluated by combining the disease activity index (DAI) score and histopathological score. DAI score was assessed for each animal as a cumulative score for the severity of colitis, according to the stool consistency (score: 0, normal; 1, soft and shaped; 2, loose stools; 3, diarrhea), rectal bleeding (score: 0, normal; 1 and 2, bloody stool; 3, gross bleeding), and body weight loss (score: 0, none; 1, 1%–5%; 2, 5%–10%; 3, 10%–15%; 4, >15%). Histopathological changes were analyzed as a cumulative score, based on epithelial damage (0, normal morphology; 1, loss of goblet cells; 2, loss of goblet cells in large areas; 3, loss of crypts; 4, loss of crypts in large areas), and inflammatory cell infiltration (0, no infiltration; 1, infiltration around crypt bases; 2, infiltration spreading to muscularis mucosa; 3, extensive infiltration in the muscularis mucosa with abundant edema; 4, infiltration spreading to submucosa).

After fixing in 10% formalin for 24 h, renal tissues were embedded in paraffin, cut into slices, deparaffinized with xylene baths, and rehydrated with graded alcohols. Hematoxylin and eosin (H&E) staining was performed as follows: (1) hematoxylin staining for 5 min at 37 °C and (2) 0.5% eosin for 2 min. Masson’s trichrome staining was performed according to the manufacturer’s protocol (Leagene, Beijing, China). Myofibers are shown in red, while collagen fibers are indicated by light green or aniline blue staining. Images were captured using an optical microscope (Leica).