Sc 515032

Cells were lysed with lysis buffer with a 1% protease inhibitor cocktail to harvest total cellular protein. The concentration of extracted protein was quantified by bicinchoninic acid (BCA) protein assay kit (Beyotime). An equal amount of protein sample was loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). After the membrane was blocked with skimmed milk, it was incubated with primary antibodies against Muc2 (Santa Cruz, sc-515032), PPARγ (Abcam, ab59256), A_2AR (Abcam, ab3461), and GAPDH (Abcam, ab8245). The next day, secondary antibodies conjugated with Horseradish peroxidase were probed for 2–3 h. Protein signals were detected with enhanced ECL chemiluminescence reagent based on the manufacturer’s instructions.

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.