Immunohistology and autoradiography
All procedures in the mice were approved by the Danish National Animal Experiments Inspectorate (license no. 2018-15-0201-01397). All mice were kept in the animal facility and received tap water and standard chow ad libitum.
Surgical procedure. Female C57BL/6JRj mice (n=9) weighing 18-26 g were purchased from Janvier (Saint Berthevin Cedex, France) and left to acclimatize for at least one week before experimental procedures were performed. The mice were anaesthetized with an intraperitoneal injection of ketamine-xylazine (100:10 mg/kg, Pharma service SUND, UCPH, Copenhagen, Denmark). The abdomen was opened by a midline incision, the inferior caval vein exposed, and [125I]-hGLP-2(1-33,M10Y) (2 pmol (3 mill cpm)) dissolved in 100 µL of 0.04 M phosphate buffer containing in addition 1% HSA (pH 7.5) was injected slowly. Three of the animals also received a 1000-fold excess of unlabeled hGLP-2(1-33,M10Y) (2 nmol) in combination with the labelled peptide in the same injection to test for specific binding. The actual amount of [125I]-labelled peptide administered was calculated from the specific radioactivity of the radioligand. Before injection, 10 µL of the [125I]-labelled peptide stock solution was counted in a gamma-counter to determine the amount of radioactivity injected into the animals. Ten min after peptide injection, the thorax was opened, after which the vascular system was perfused at a constant flow with 0.9% saline with an outlet through the right ventricle. Next, the mice were fixated by flushing the system with ice-cold 4% paraformaldehyde. After fixation, the pancreas, small intestine and kidneys (as positive control) were removed and stored in 45% paraformaldehyde until further processing.
Autoradiography. Small intestinal, pancreatic and kidney tissue samples were embedded in paraffin, and histological 4 µm sections were cut with a microtome and placed on glass slides. The sections were dewaxed and coated in a dark room with 43-45oC Kodak NTB emulsion (VWR, Herlev, Denmark) diluted 1:1 with 43-45oC water, and subsequently dried and stored in light-proof boxes at 5oC for 6 weeks. After 6 weeks, the tissue sections were developed in a dark room in Kodak D-19 developer (VWR, Herlev, Denmark) for 5 min, dipped 10 times in 0.5% acetic acid, and fixated in 30% sodium thiosulphate for 10 min. The sections were then washed, first in water for 10 min and then in 70% ethanol. Finally, the sections were lightly counterstained with haematoxylin and examined with a light microscope (Orthoplan, leitz). Images were taken with an AxioCam ICc5 camera (Zeiss) connected to the light microscope.