Glucagon

Mice were fasted for 2 h and were injected intraperitoneally with 0.75 units/kg Humulin R insulin as previously described [19 (link)]. Islet histology procedures have been described [19 (link), 20 (link)]. Briefly, the antibodies used were as follows: insulin (Invitrogen; 18-0067; used at 1 : 800), glucagon (Cell Signaling Technologies; 8233; used at 1 : 400), Aldh1a3 (Novus Biologicals; NBP2-15339; used at 1 : 100), and Nkx6.1 (F55A12; 1 : 200; [21 (link)]). All antibodies were incubated overnight at 4°C and detected with either Alexa Fluor secondary conjugation (Alexa 488, 594), except for Nkx6.1, which was detected using a biotin/streptavidin exposure method. Substrate oxidation was measured in the liver, and gastrocnemius muscle homogenates were prepared and used as described previously [22 (link), 23 (link)]. Briefly, using [1-¹⁴C] palmitate (100 μM), both complete (¹⁴CO₂) and incomplete (¹⁴C-acid soluble metabolites (ASMs)) fatty acid oxidation were assessed ± varying doses of unlabeled pyruvate (0, 0.1, 1, and 10 mM) to assess metabolic flexibility.

Following fixation in 10% NBF for 24–48 h, pancreatic tissue was embedded in paraffin, sectioned, stained, and analyzed as previously described [20 (link),21 (link)]. Primary antibodies used were as follows: guinea pig anti-insulin (Invitrogen, Grand Island, NY, USA; #18-0067; 1:800), glucagon (Cell Signaling Technology, Danvers, MA, USA; #2760; 1:300), Nkx6.1 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA; #F55A12; 1:100); and Aldh1a3 (Novus Biologicals, Centennial, CO, USA; #NBP2-15339; 1:100).

Immunohistochemical analysis of Snail1 protein in normal and neoplasic pancreas was performed as previously described by using mAb EC3 [8 (link), 14 (link)], using 4 μm sections. For antigen unmasking, sections were immersed in Tris-EDTA buffer (pH 9) and boiled for 20 min. Immunohistochemical staining was carried out with anti-Snail1 MAb EC3 supernatant at 1/300 (murine) or 1/100 (human) dilution using the CSAII Amplification System (Dako, Glostrup, Denmark), in a Dako Link platform. Human samples (six PanIN and five pancreas adenocarcinomas) were obtained from Parc de Salut MAR Biobank (MARBiobanc), Barcelona. The analyses were approved by the Ethical Committee for Clinical Research of PRBB (Barcelona) and informed consent was obtained from all subjects.
Other proteins were analyzed with the following antibodies: desmin, GFAP (both from DakoCytomation), β-catenin (Sigma Aldrich or Transduction Labs), amylase (Sigma-Aldrich), CK19 (Abcam), CD105 (Abgent), S100A4 (Millipore), insulin, glucagon, and active caspase-3 (Cell Signalling). Sections were counterstained with hematoxylin.