Immunofluorescence

Isolated human islets cultured on ECM dishes were fixed in Bouin’s solution for 15 min before embedding in paraffin as previously described (14). 4-µm sections were deparaffinized, rehydrated and incubated overnight at 4°C with primary antibodies against hGLP-2R (1:100; LS-A1312), hGLP-2R (1:100, {"type":"entrez-nucleotide","attrs":{"text":"C36446","term_id":"2372587","term_text":"C36446"}}C36446, both Lifespan Biosciences, Seattle, WA), guinea pig anti-insulin (#A0546, 1:100, Dako, Glostrup, Denmark) or mouse anti-glucagon (#G2654, 1:100, Sigma-Aldrich, Steinheim, Germany), followed by incubation with FITC or Cy3-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA) at room temperature for 1h. Slides were mounted with Vectashield with DAPI (Vector Labs, Burlingame, Ca). Fluorescence was analyzed using a Nikon MEA53200 microscope (Nikon GmbH, Dusseldorf, Germany) and images were acquired using NIS-Elements software (Nikon).