Ab256345

After the mice of each group were sacrificed, the proximal colon was collected immediately, fixed with 10% formalin, embedded in paraffin, sectioned to a thickness of 5 mm, deparaffinized and submitted to hematoxylin and eosin (H&E, Sigma-Aldrich, Shanghai, China) staining. Immunohistochemistry was performed using a previously described method (Huang et al., 2019 (link)). Briefly, 3 μm sections were deparaffinized in xylene and rehydrated in graded alcohol. After quenching endogenous peroxidase activity and blocking non-specific binding, the sections were incubated with a rabbit monoclonal antibody [EPR22566-344] against c-Kit (Abcam, ab256345) overnight at 4°C and then incubated with the secondary antibody goat anti-rabbit IgG (H + L) HRP (ab0101, Abways) at room temperature for 30 min. Finally, the slides were incubated with reagents from the Avidin-Biotin Complex Kit (Vector Laboratories, Inc., Burlingame, USA) and a 3,3′-diaminobenzidine kit (Tiangen, China) according to the manufacturer’s instructions. Images were captured with a Nikon Ci-S microscope and Nikon DS-U3 imaging system. The proportion of c-Kit-positive cells in the colonic muscle layer was determined using an image analyzer (Image-Pro Plus 6.0, Media Cybernetics, Inc., Rockville, USA).

Total proteins were extracted from mouse tissues or cells using RIPA Lysis Buffer (#P0013B; Beyotime), following the manufacturer's instructions. A total of 30 μg of protein from each sample were boiled at 100°C for 5 minutes, separated through SDS‐PAGE, and transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% lipid‐free milk for 1‐2 hours, incubated with primary antibodies diluted in TBST for 2 hours at room temperature, washed with TBST and incubated with secondary antibodies for 1 hour at room temperature. Protein abundances were determined by development with enhanced chemiluminescence (ECL) substrates (Millipore). GAPDH was used as an internal standard. Primary antibodies targeting c‐Kit (#ab256345; Abcam), P65 (#ab16502; Abcam), p‐P65 (#ab86299; Abcam), AKT (#9272; CST), p‐AKT (#4060; CST) and GAPDH (#ab8245; Abcam) were applied.