Immunohistochemical Analysis of Gut Hormones

For the immunohistochemical study, the tissue sections after dewaxing and rehydration were heated for 15 min in citrate buffer (0.01 mol/L, pH = 6.0) to recover the antigens. Subsequently, the tissue sections were placed in 3% H₂O₂ for 15 min to incubate, and then washed with phosphate-buffered saline (PBS, 0.01 mol/L). Afterward, the tissue sections were incubated in goat serum blocking solution at room temperature for 30 min, and then incubated overnight at 4 °C with the primary antisera (anti-serotonin, anti-glucagon, and anti-gastrin; dilution ratio = 1:200). Then, the sections were incubated with a biotinylated secondary antibody, followed by staining with DAB colorimetric solution (dilution ratio = 1:20) at room temperature and re-staining with hematoxylin staining solution [18 (link),31 (link)]. Finally, the sections were subjected to dehydration, immersed in a clearing agent to enhance their transparency, and sealed with neutral gum. All of the above reagents were from Beijing Zhongshan Golden Bridge Biological Company, China. Three microphotographs (400× magnification) were randomly acquired from each part of each sample by using a Digital trinocular microscope camera system (BA400Digital, Motic, Xiamen, China). The distribution characteristics of 5-HT-immunoreactive cells, Gas-immunoreactive cells, and Glu-immunoreactive cells were observed and their positive expression levels (the proportion of DAB positive tissue = DAB positive area/tissue area × 100%) were calculated using the Data Image Analysis System (Halo 101-WL-HALO-1, Indica labs, Albuquerque, NM, USA).