Cryopreservation of Mouse Intestinal Organs

Ten mice were bred from a C57BL/6 background (5 WT AQP1^+/+, 4 KO AQP1^−/− and 1 HT AQP1^−/+) and the intestines provided by from the Department of Physiology of the Hannover Medical School (Hannover, Germany). The AQP1-KO mice had been generated from breeding pairs of heterozygous AQP^−/+ mice kindly provided by Dr. Alan S. Verkman (San Francisco, CA, USA; Ma et al., 1998). Animals were anaesthetized and sacrificed in accordance with the German Tierschutzgesetz §4 2015/222. Organs, specifically the esophagus, stomach, duodenum, jejunum, ileum, cecum, and colon were immediately removed. These organs were separately snap frozen and cryo-conserved. Each organ was embedded in OCT (TissueTek; Thermofisher Scientific Inc., Waltham, MA, USA) according to a modification of the “Swiss role” described by Meier-Ruge [65 ], with exception of the esophagus, which was embedded as a tube for cryo-cutting. The samples were cut with the cryotome (Leica CM1950) into 7 µm slices, fixed on a microscope slide and stored at minus 20 °C.