Quetol 812

For electron microscopy, 8–12-wk-old control, AcKO, or PcKO mice were used. CCK (0.25 µg/kg weight) or glucose (1 g/kg weight) was administered by i.p. injection. 30 min after the injections, the mice were perfused intracardially with 2.5% glutaraldehyde and 2% PFA in 0.1 M cacodylate buffer, pH 7.4. The pancreas was removed from each mouse and fixed in the same fixative and 0.1% OsO₄, after which it was further fixed in 0.5% uranyl acetate in H₂O, dehydrated, and embedded in Quetol 812 (Nisshin EM). Ultrathin sections were cut using an ultra-microtome (Reichert Jung). Electron micrographs were taken using a JEM-1010 (JEOL).

Electron microscopy was performed by Tokai Electron Microscopy, Inc. (Nagoya, Japan). Tissue samples of approximately 2 mm³ were resected from the vicinity of the glandular stomach–jejunal anastomosis, a site of frequent localization of PACM, from rats 25 weeks postoperatively, while tissues of the glandular stomach mucosa and pancreas were resected from the control rats at 25 weeks of age. The cells were permeabilized and fixed in 2% paraformaldehyde and 2% glutaraldehyde. After washing, cells were fixed with 2% osmium tetroxide followed by dehydration with alcohol. Infiltration with propylene oxide and resin embedding (Quetol-812; Nisshin EM Co., Tokyo, Japan) were performed. The slices were further cut into 1.5 μm sections with a glass knife and stained with 0.5% toluidine blue. Ultra-thin slicing was performed at a thickness of 70 nm using a diamond knife. The primary staining was performed with 2% uranyl acetate and the secondary staining was performed with a lead stain solution (Sigma-Aldrich Co., Tokyo, Japan). Stained sections were observed using a transmission electron microscope (JEM-1400Plus; JOEL Ltd., Tokyo, Japan), and digital images were acquired using a CCD camera (3296 × 2472 pixels, EM-14830RUBY2; JEOL Ltd., Tokyo, Japan).

Electron microscopy was performed by Tokai Electron Microscopy Inc. The samples were fixed with 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), followed by postfixation with 2% osmium tetroxide. The fixed samples were dehydrated through a series of ethanol concentrations (50, 70, 90, and 100%) and then transferred to a resin (Quetol-812; Nisshin EM Co., Tokyo, Japan) and polymerized at 60°C.
The blocks were ultrathin sectioned at 70 nm using an ultramicrotome (Ultracut UCT; Leica) and stained with 2% uranyl acetate followed by lead stain solution (Sigma-Aldrich). The samples were observed by a transmission electron microscope (JEM-1400Plus; JEOL Ltd.) at an acceleration voltage of 80 kV. Digital images (2048 × 2048 pixels) were captured with a charge-coupled device camera (VRLRTA; Olympus Soft Imaging Solution GmbH).