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.