Oct cryocompound tissue freezing medium

After removal of the storage compartment fundus, the stomach was opened along the greater curvature and subdivided along the lesser curvature into two equal halves, each further separated into corpus and antrum in 1 × phosphate-buffered saline (PBS) (0.85% NaCl, 1.4 mM KH₂PO₄, 8 mM Na₂HPO₄, pH 7.4). For RNA isolation material was immediately transferred into a collection tube, frozen in liquid nitrogen and stored at −70°C until use and for immunohistochemical analyses immersion-fixed in 4% buffered paraformaldehyde in 150 mM phosphate buffer, pH 7.4) for either 15 min (for GPR120, TRPM5, ghrelin and gastrin) or 4 h (for GPR43) at 4°C followed by cryoprotection in 25% sucrose at 4°C overnight. Subsequently, gastric tissue was embedded in Leica OCT cryocompound tissue freezing medium (Leica Microsystems, Bensheim, Germany) and quickly frozen on liquid nitrogen. Longitudinal sections (8 μm) were cut on a CM3000 cryostat (Leica Microsystems, Bensheim, Germany) and attached to Superfrost Plus microslides (Menzel Gläser, Braunschweig, Germany).

After removal of the storage compartment fundus, the stomach was opened along the greater curvature and washed in 1 × phosphate-buffered saline (PBS: 0.85% NaCl, 1.4 mM KH₂PO₄, 8 mM Na₂HPO₄, pH 7.4). For RNA isolation, the dissected corpus was immediately transferred into a collection tube, frozen in liquid nitrogen, and stored at −70°C until use. For immunohistochemical analyses, the dissected stomach was immersion-fixed in 4% buffered paraformaldehyde in 150 mM phosphate buffer (pH 7.4) for 2 h at 4°C followed by cryoprotection in 25% sucrose at 4°C overnight, then embedded in Leica OCT cryocompound tissue freezing medium (Leica Microsystems, Bensheim, Germany), and quickly frozen on liquid nitrogen. Longitudinal sections (4–8 μm) were cut on a CM3000 cryostat (Leica Microsystems, Bensheim, Germany) and collected on Superfrost Plus microslides (Menzel Gläser, Braunschweig, Germany).