G1285

Alcian blue and periodic acid-Schiff (AB-PAS) staining were performed with the panel (Solarbio, G1285). The prepared sections were treated with Alcian blue dye for 5 min after deparaffinization and rehydration, washed with running water for 2 min, and stained with periodic acid for 5 min. The slices were treated for 30 min with Schiff’s stain under protection from light. After washing with running water, the sections were subjected to hematoxylin staining, followed by dehydration through xylene. Images were obtained under a computer-supported imaging system connected to a light microscope.

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)].

For each mouse, three sections were selected to be stained with Alcian Blue Periodic acid Schiff (AB/PAS) and HE staining. AB/PAS staining was performed using the panel (Solarbio, G1285). The prepared sections were treated with Alcian blue dye for 10 min following deparaffinization and rehydration, then washed with running water for 5 min, and subsequently stained with periodic acid for 3 min. The sections were oxidized and washed twice, followed by staining with Schiff’s reagent for 12 min. After a 10-min rinse in distilled water, the sections underwent hematoxylin staining. They were then treated with Scott’s Bluing Solution for 3 min and washed for an additional 3 min. Finally, the sections were dehydrated using a series of ethanol washes and cleared with xylene before sealing. The goblet cell was observed using AB/PAS staining.
The colon tissue was fixed in 10% neutral buffered formalin for 24 h, subsequently dehydrated, and then embedded in paraffin wax. Approximately 5 μm sections of the paraffin-embedded tissue were cut and stained with HE (Solarbio, G1120). The goblet cells are round cells that appear clear on HE staining. All the sections were observed and photographed under a light microscope (Olympus, Tokyo, Japan).

The cecum and cecal contents were collected at the time of sacrifice. Intestinal mucins were detected by Alcian blue and periodic acid-Schiff (AB/PAS) staining (G1285, Solarbio). Following the manufacturer's instructions, AB staining was performed first (at pH 2.5) for 10–20 min, followed by periodic acid-Schiff staining. Fecal lactate levels were measured by a colorimetric assay kit (A019-2-2, Jiancheng). The fecal wet-to-dry ratio was defined as (wet weight–dry weight)/dry weight. Fecal noradrenaline and adrenaline levels were measured by targeted high-performance liquid chromatography mass spectrometry (HPLC-MS; AB ExionLC, AB Sciex Qtrap 6500+, AB Sciex).