Anti chat

After the animals were euthanized, the antrum was collected and stored immediately at −80°C. Total protein was extracted from gastric antrum homogenate, and the concentration of protein was determined using an enzyme-linked immunosorbent assay at 562 nm. The protein sample was boiled, and then equivalent amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 8% or 12% gels. The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. TBS-T buffer (Tris buffer saline, 0.1% Tween) containing 5% BSA was used to block the membrane at room temperature for 60 minutes. Then, the membrane was incubated with the primary antibodies at 4°C overnight. Horseradish peroxidase (HRP) conjugated secondary antibodies were then incubated with the membranes at room temperature for 60 minutes. The gray values of the immunoreactive protein bands were quantified using ImageJ software (NIH, Bethesda, MD, USA). The antibodies used included anti-PGP9.5 (rabbit, 1 : 1000, Abcam, Cambridge, UK), anti-nNOS (rabbit, 1 : 1000, Abcam), anti-ChAT (rabbit 1 : 1000, Abcam), anti-GAPDH (1 : 1000, Cell signaling Technology, Danvers, MA, USA), and anti-Vinculin (1 : 2000, Abcam, Cambridge, UK) antibodies.

Cells were washed once with 1 × PBS and fixed in 4% paraformaldehyde (Leagene) for 15 min at room temperature. After washing three times with 1 × PBS, cells were permeabilized with 0.5% Triton X-100 in 1 × PBS for 15 min, washed three times with 1 × PBS, and blocked with 10% goat serum for at least 30 min. Then, all primary antibodies were diluted in 1 × PBS and incubated cells overnight at 4°C. Primary antibodies were anti-Nestin (Millipore, 1:100, rat), anti-Sox2 (abcam, 1:100, mouse), anti-Tuj1 (Santa cruz, 1:100, mouse), anti-NeuN (abcam, 1:300, rabbit), anti-GFAP (Santa cruz, 1:100, rabbit), anti-olig2 (Proteintech, 1:1000, rabbit), anti-TH (Santa cruz, 1:100, rabbit), anti-ChAT (abcam, 1:100, rabbit), anti-GABA (sigma, 1:100, mouse), anti-Nav 1.7 (abcam, 1:100, mouse), anti-COL1A1 (Bioss, 1:400, mouse), anti-Collagen IV (abcam, 1:250, rabbit), and anti-Oct4 (Bioss, 1:400, rabbit). Next, cells were washed three times with 1 × PBS and incubated with secondary antibodies FITC (bioss, 1:100) or Cy3 (cwbio, 1:100) for 30 min at room temperature, followed three 5 min-washed with 1 × PBS. The nuclei were stained with DAPI (BestBio, 1:100) for 15 min.