3 3 diaminobenzidine kit

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).

Immunohistochemistry was performed using a previously described method (17 (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 mouse monoclonal antibody against CD177 or F4/80 (Santa Cruz, CA, USA) overnight at 4°C and then incubated with a biotinylated secondary antibody (Zymed Laboratories, Carlsbad, CA, USA) at room temperature for 20 min. Finally, the slides were incubated with reagents from the Avidin-Biotin Complex Kit (Vector Laboratories, Burlingame, CA, USA) and a 3,3'-diaminobenzidine kit (Tiangen, Sichuan, China) according to the manufacturer's instructions. Images were captured with an Olympus CX43 microscope and CellSens Entry software.