Ab6336

At the end of the 12-week experimental period, mice were deeply anesthetized with 4% tribromoethanol and perfused intracardially with 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS, pH 7.4). Spinal C5–C7 segments and biceps (six animals in each group) were collected and post-fixed in the same fixative overnight, and then immersed in 10%-20%-30% sucrose until they sank. Ten micrometer transverse spinal sections and 40-μm horizontal biceps sections were prepared with a sliding microtome (Leica). Sections were kept in 12-well plates at 4°C. After washing with PBS, sections were blocked in 10% goat serum plus 3% bovine serum albumin for 2 h, and incubated with the primary antibodies overnight at 4°C. The primary antibodies included: goat anti-choline acetyltransferase (anti-ChAT; 1:500; AB144p, Millipore), rabbit anti-oligodendrocyte transcription factor 2 (anti-Olig2; 1:2,000, AB9610, Millipore), mouse anti-serotonin (anti-5-HT; 1:500, ab6336, Abcam) and rabbit anti-tyrosine hydroxylase (anti-TH; 1:500; AB152, Millipore). Sections were rinsed in 0.01 M PBS and incubated with the secondary fluorescent antibodies (Alexa Fluor 488 or 546; 1:1,000, A21202/A21206/A11055, Thermo Fisher) for 2 h. α-bungarotoxin conjugated to Alexa Fluor 546 (α-BT; 1:1,000, T1175, Molecular Probes) was used to label acetylcholine receptor clusters.

The colon sections were incubated with anti-5-HT (ab6336, Abcam, United States) and anti-VIP (ab30680, Abcam, United States) antibodies (dilution 1:100) for 24 h at 4°C. Subsequently, the immunoreaction was revealed by using HRP-labeled secondary goat anti-rat antibody (ab97057, Abcam, United States, dilution 1:500) for 2 h at room temperature. Then the slides were stained with 3,3′-Diaminobenzidine (DAB) substrate solution, and counterstained with Hematoxylin. The slides were scanned using a microscope (Olympus, Tokyo, Japan). Five high-power fields (×400 magnification) from each slide were randomly selected and analyzed using Image-Pro Plus image analysis software. Integrated option density (IOD) and area value of each image were measured, and then mean density (mean density = IOD/area) was calculated. The mean optical density values were used to plot the relative expression levels of the proteins.

The embedded tissues were baked for 20 min at 60 °C, then dewaxed and hydrated as described in HE staining. The sections were placed in 0.01 mol/L sodium citrate buffer (pH = 6.0) and heat them boiling using microwave oven (repeating 2 times at an interval of 10 min). After washing with 0.01 mol/L PBS (pH = 6.0) for 3 times, 5 min each time, the sections were cultured with 3% H₂O₂ for 10 min, then washed with PBS for 3 times. The primary antibodies including α2δ2 (#PA5-77341, 1:400, ThermoFisher Science, China), 5 hydroxytryptamine (5HT, #ab6336, 1:1000, Abcam, China), GAP43 (#ab277627, 1:1000, Abcam, China), Iba-1 (#ab178847, 1:100, Abcam, China) were added to the sections and cultured for overnight at 4 °C. After washing with PBS for 3 times, the goat secondary antibody goat (1:500, ThermoFisher Science, China) were added to culture for 60 min at 37 °C. Then, the sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min. After washing with PBS and quenching fluorescence, the sections were observed under a laser confocal microscope (LSM800, Zeiss, Germany).