Ab pas

Paraffin-embedded tissue samples were sectioned into 3 um thin slices. The TUNEL of apoptotic cells in mouse colon tissue was detected using a One Step TUNEL Apoptosis Assay Kit (#C1088, Beyotime), following the manufacturer’s protocol. TUNEL-positive cells were visualized under fluorescent microscopy to evaluate apoptotic and necrotic morphological alterations. AB-PAS (#G1285, Solarbio, Beijing, China) staining was performed in accordance with the manufacturer’s instructions.

Colon tissues were fixed in 4% paraformaldehyde in PBS, embedded in paraffin, sectioned, and stained with AB-PAS (Solarbio, G1285). Images of the sections were captured to count the number of goblet cells per crypt.

Ileum tissue was taken to prepare paraffin sections. The prepared paraffin sections were baked and dried, and haematoxylin and eosin were used for HE staining. The stained sections were digitally captured with an integrated fluorescence microscope, and 3–5 complete villus structures in each field were measured using ImageJ software. According to the instructions of the Alcian blue-periodic acid-Schiff (AB-PAS) staining kit (#G1285; Solarbio, China), after 10 minutes of oxidation with periodic acid, Schiff reagent was added for staining. The stained sections were digitally captured using an integrated fluorescence microscope, and the number of goblet cells per unit length was measured using ImageJ software.

Samples were fixed in 4% paraformaldehyde solution and embedded in paraffin. Then, 5 μm sections were made and mounted on slides for staining with hematoxylin and eosin (H&E, G1120, Solarbio, Beijing, China), Masson (G1340, Solarbio, Beijing, China), Sirius Red (G3632, Solarbio, Beijing, China) and AB-PAS (G1285, Solarbio, Beijing, China), according to the manufactures’ instructions. For Oil Red O staining, frozen sections of the livers were first obtained, and the staining was performed according to the manufacturer’s instructions (G1260, Solarbio, Beijing, China), while the quantification was performed according to a previous protocol [91 (link)].
With H&E staining, hepatocellular steatosis was graded from 0 to 3 based on the percentage of hepatocytes involved (0 = <5%; 1 = 5–33%; 2 = 33–66%; 3 = >66%), according to a previous study [92 (link)]. The NAS score was calculated by steatosis (0–3), lobular inflammation (0−3) and ballooning (0−2), also according to a previous study [32 (link)].

Organ samples were fixed in 4% paraformaldehyde solution for more than 48 h, then embedded in paraffin. Paraffin sections with a thickness of 5 μm were made and mounted on slides for staining with hematoxylin and eosin (H&E, G1120, Solarbio, Beijing, China) and AB-PAS (G1285, Solarbio, Beijing, China), according to the manufactures’ instructions.