Nb100 92156

The protocol was largely as previously described [28 ]. Briefly, the detached epithelial cell lysates were mixed with 2X modified Laemmli Sample Buffer containing 8 M urea and incubated for 30 min at room temperature. Following centrifugation (2 min, 8,000 x g), samples of the supernatant (adjusted to 40–50 μg protein) were separated on a 6 % polyacrylamide gel. Immunoblot analysis was performed with an anti-CFTR antibody (NB100-92156, Novus Biologicals), followed by incubation with a secondary antibody conjugated with horseradish peroxidase. Peroxidase activity was detected by incubating with Luminata Forte Western HRP substrate (Millipore) and exposure to X-ray film (Kodak® BioMax® XAR Film).

Briefly, the duodenum, defined as the first 3 cm of the small intestine, was dissected from wild type, untreated CF and CF mice treated by whole body cooling. The duodenum was fixed with 4 % paraformaldehyde solution and then embedded in paraffin before being sectioned. Paraffin-embedded mouse small intestine was sectioned in 5 μm thick slices. For anti-CFTR staining, sections were pretreated by heat-induced epitope retrieval, blocked in 10 % goat serum for 1 h and then incubated overnight with primary antibodies for CFTR (NB100-92156, Novus Biologicals) at 1:100 in incubation medium (PBS with 3 % goat serum, 3 % BSA, and 0.3 % Triton × 100). After four 5 min washes in washing buffer, the sections were incubated with secondary antibodies (Alexa Fluor 555–labeled goat anti-rabbit IgG; Invitrogen) for 1 h at room temperature at a concentration of 2 μg/ml in incubation medium. After four washes for five min each, cover slips were mounted with SlowFade Gold antifade reagent with DAPI (Invitrogen). Some slides were co-stained with wheat germ agglutinin, Alexa Fluor® 488 conjugate (Invitrogen), to visualize mucin secreted by goblet cells in mouse intestine.