Isolation of Mouse Gut Muscularis Externa

The muscularis externa containing myenteric plexuses was prepared from mouse GITs as previously described with some modification (65 (link), 66 (link)). For HILIC-MS/MS analysis, mouse GIT segments were cut open along the attachment line of the mesentery and then placed onto a cold surface with the muscularis externa facing up. The muscularis externa of the GIT segments was isolated by gently scraping the outer layer with watchmaker tweezers under a binocular stereomicroscope. For whole-mount immunostaining, the mouse duodenum was cut open along the mesentery line, pinned onto a rubber plate, and then fixed with 4% paraformaldehyde overnight at 4 °C. The muscularis layer was then gently separated from the GIT segment using watchmaker tweezers and a cotton swab under a binocular stereomicroscope. For RNA extraction, mouse GITs were immersed in saturated ammonium sulfate solution containing 20 mM EDTA and 25 mM sodium citrate (pH5.2) to inhibit RNA degradation. The muscularis externa of the segments was placed over a glass rod and then peeled away using a cotton swab along the attachment line of the mesentery under a binocular stereomicroscope as described (67 ). Isolated muscularis externa was stored in saturated ammonium sulfate solution and then frozen until further use.