Processing of the Pancreatic Specimens for TEM

The pancreatic samples were processed in plastic-ware as follows: 25 mg of tissue was minced and digested overnight at ambient temperature with 1 mL of an aqueous solution containing 25 mg of trypsin (from bovine pancreas, Sigma catalog number 1426) and 25 mg of proteinase K (from Tritirachium album, Sigma catalog number P2308). The proteolytic enzyme-treated suspensions were degreased with hexane. First the suspensions were centrifuged at 12,000g for 1 h to separate an oily top layer from the aqueous phase. The oily layer solidified upon chilling to 0 °C, allowing removal of most of the oil by skimming with a 2 mm wooden applicator. Because part of the aqueous suspension was emulsified in the skimmed oil and it could have contained crystals, the oil was dissolved in 1 mL of hexane and centrifuged for 1 h at 12,000g. The top hexane layer was pipetted off, and the bottom aqueous layer was re-extracted with 0.5 mL hexane and centrifuged for 20 min at 12,000g. After the hexane was pipetted off, the aqueous centrifugate was combined with the main aqueous suspension. The combined hexane phases were re-extracted with 0.5 mL of water and centrifuged for 20 min at 12,000g, and the aqueous centrifugate was again combined with the main aqueous suspension. The aqueous suspension was recentrifuged at 12,000g for 10 min to separate residual hexane. About 1 mL of the now degreased aqueous suspension was obtained. After shaking to homogenize, 5 μL of the aqueous suspension was water diluted 200-fold to 1 mL. Although the diluted aqueous solution appeared to be clear and homogeneous, it was shaken to homogenize, then 5 μL of the solution was pipetted onto the 3 mm diameter TEM grid. The liquid on the grid was evenly distributed, and the examined squares were selected from both centers and peripheries of the grids.