Ab pas stain kit

To make paraffin sections, the colon and Mes-WAT were fixed in Carnoy’s solution for 2 h or 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 µm following hematoxylin and eosin (H&E) and alcian blue periodic acid Schiff (AB-PAS) staining. H&E staining was performed according to standard methods as we previously used [107 (link)], briefly, hematoxylin solution for 3 min and eosin solution for 1 min. For the histological score of colitis, H&E staining colon sections were scored by an individual blinded to the details using a previously published system as indicated [106 (link)] (range 0–3), including crypt architecture, degree of inflammatory cell infiltration, muscle thickening, and goblet cell depletion. The histological score of colitis is the sum of each mouse. For AB-PAS staining, sections were stained with an AB-PAS stain kit (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China).

Colon tissues were embedded in paraffin, and serial 5-μm-thick sections were placed onto slides. After dewaxing and rehydration, the sections were stained with H&E according to routine protocols. For AB-PAS staining, colon sections were stained using the Alcian Blue Periodic Acid Schiff (AB-PAS) Stain Kit (G1285, Solarbio).

The mucus secretion capacity of the mice was analyzed by AB-PAS Stain kit (Beijing Solarbio Science & Technology Co., Ltd.) following the manufacturer's guidelines. After dewaxing, the tissue sections were stained with Alcian blue dye solution and then washed in running water. Second, the sections were stained with 0.5% periodic acid solution for 15 mins and washed twice with distilled water. Third, the tissues were soaked in Schiff reagent under the dark conditions for 30 mins and then rinsed for 5 mins. The slides were dehydrated with gradient alcohol and cleared with xylene to be transparent. The sections were observed for the mucus layer and goblet cells under an optical microscope (Nikon Italia, Italy). At least 20 randomly selected fields were assessed to quantify goblet cells under × 400 magnification, and data were averaged for each colon (n = 8).

For histological analysis, distal colon tissue was fixed in 4% paraformaldehyde and embedded into paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin. Images were captured with a microscope (BX46, Olympus, Tokyo, Japan). Histological score was evaluated according to the criteria described before [37 (link)]. Briefly, histological score was scored with seven parameters: degree of inflammation (0–4), the severity of crypt damage (0–4), immune cell infiltration (0–3), submucosal edema (0–3), loss of goblet cells (0–3), active epithelial hyperplasia (0–3), and crypt abscesses (0–2).
For goblet cell detection, AB-PAS staining was performed with the AB-PAS Stain Kit (Solarbio, Beijing, China) according to the manufacturer’s recommendations. Images were captured with a microscope (BX46, Olympus). The total number of goblet cells was counted in crypts from 4 different fields.

Mice were sacrificed at different ages (12 w, 25 w, and 35 w). Mouse stomachs were then harvested, cut open through the greater curvature, and washed 3 times in phosphate-buffered saline by vigorous shaking. Tissues were fixed in 4% poly-formaldehyde for 24 hours at 4°C, then dehydrated and embedded in paraffin. Sections (5 μm) were cut and stained with H&E. Tumor grades (0-4) were scored according to the previous report [39 ]. For IHC staining, sections were deparaffinized, rehydrated, subjected to antigen retrieval in citrate buffer, and quenched for endogenous peroxidases with 3% H₂O₂. Blocking was performed with 5% BSA for 1 hour at room temperature. The primary antibodies used here were anti-c-MYC (Abcam, ab32072, 1:200), anti-Ki67 (Abcam, ab15580, 1:2000), anti-MUC2 (Santa, sc-515032, 1:200), and anti-MUC5AC (Abcam, ab3649, 1:200). Periodic Acid-Schiff/Alcian Blue (PAS/AB) staining was performed using AB/PAS stain kit (Solarbio, G1285). Staining intensities were calculated using Image J software.