Sylgard lined petri dish

At the time of resection, a 2 cm ring of tissue was cut from the uninvolved healthy margin of the colonic specimen. The colonic tissue was transported in oxygenated Krebs solution (pH; 7.4, NaCl; 118 mM, KCl; 4.8 mM, CaCl₂; 2.5 mM, MgSO₄; 1.2 mM, NaHCO₃; 25 mM, NaH₂PO₄; 1.0 mM, glucose; 11 mM, bubbled with 95% O₂ and 5% CO₂) from the operating theatre to a laboratory in the same building. The colonic tissue was pinned out in a Sylgard-lined petri dish (Dow Corning, Midland, MI, United States) under a dissecting microscope. Oxygenated Krebs solution was regularly changed. The mucosa, submucosa and some of the serosa were removed by microdissection. The tissue was then stretched maximally in both longitudinal and circular axes and fixed with 4% paraformaldehyde (pH 7.2, 4% paraformaldehyde in 0.1 M phosphate buffer) overnight at 4°C. It was then further fixed in 4% paraformaldehyde on an orbital mixer at room temperature, for a total of 24 h, thus ensuring penetration of the fixative. Subsequently the tissue was further dissected in 1× phosphate buffered saline (PBS) to remove most of the circular muscle and any remaining submucosa and serosa. This produced a wholemount of longitudinal muscle and myenteric plexus, from which a 10 mm × 15 mm specimen was cut from a region between the taenia coli.