Anti glucagon

Pancreas tissue was fixed in buffered 10% formalin, processed to wax blocks and sectioned onto slides (4 μm). Dual fluorescence staining was performed to determine the islet cell type in which β-galactosidase/GPR120 was expressed. Anti-insulin (1:500; Dako, Cambridge, UK), anti-glucagon (1:200; Abcam, Cambridge, UK) and anti-somatostatin (1:100; Millipore, Watford, UK) were detected using Alexa Fluor-488 conjugated anti-goat antibody (Abcam) and anti-β-galactosidase (1:1200; Abcam) with Alexa Fluor-568 conjugated anti-rabbit antibody. Citrate buffer (pH 6) was used for antigen retrieval in the case of β-galactosidase and somatostatin. Briefly, tissue sections were fixed, de-waxed and rehydrated prior to antigen retrieval (heating in a microwave at full power for 20 min followed by cooling to room temperature). The sections were blocked before the addition of primary antibodies and washed prior to the addition of secondary antibodies and DAPI nuclear stain (1:500; Thermo Scientific). Slides were mounted in Hardset Vectashield Mounting medium (Vector Laboratories, Peterborough, UK) and analysed on a Nikon Eclipse 90i Fluorescent Microscope.

The pancreas was weighed, fixed, processed, and embedded in paraffin. Whole pancreas was then sectioned with 5 µm thickness, then sections were selected at intervals of 250-300 µm for immunohistochemical staining of insulin and counterstained with eosin. The sections were then scanned using an Axio Scan.Z1 (ZEISS, Oberkochen, Germany). The proportion of insulin-positive in total pancreatic staining area versus the whole pancreas area was calculated using image J. Immunofluorescence staining images were captured by confocal microscope LSM 880 (ZEISS). Antibodies used in the study included anti-Insulin (1:800, DAKO, Copenhagen, Denmark), anti-UCN3 (1:500, Phoenix Pharmaceuticals, Mannheim, Baden-Wurttemberg, Germany), anti-Ki67 (1:400, DSHB), anti-Glucagon (1:500, Abcam), anti-Somatostatin (1:100, Abcam), and anti-ALDH1A3 (1:100, NOVUS). Fluorescence detection was operated with Alexa Fluor 647 nm, 488 nm and 594 nm conjugated antibodies (Thermo Fisher Scientific). Cell nuclei were labelled with DAPI.

Prior to immunostaining, the sections were treated sequentially with 3% hydrogen peroxide, citrate-based antigen retrieval solution (Beyotime Biotechnology, Shanghai, China) and goat serum blocking dilution. Insulin and glucagon were stained with anti-insulin (1:200; Cell Signaling Technology, Danvers, MA, U.S.A.) and anti-glucagon (1:200; Abcam, Cambridge, MA, U.S.A.) antibodies, respectively, followed by reacting with 1:1000 either Alexa Fluor 488-conjugated donkey anti-rabbit or Alexa Fluor 594-conjugated donkey anti-mouse IgG antibodies (Invitrogen, Carlsbad, CA, U.S.A.), while nuclei were visualized with 4′,6-diamidino-2-phenylidole (DAPI; Invitrogen). Sections were analyzed using a Vectra automated quantitative pathology system (PerkinElmer, Waltham, MA, U.S.A. ).

Immunofluorescence was performed as previously described.32, 33, 36, 37 The mice were sacrificed by cervical dislocation, and the pancreata were immediately dissected, washed in PBS and fixed in 4% formaldehyde at 4°C for at least 8 hours. Next, the samples were kept in different concentration of sucrose (4 hours in 10% sucrose, 8 hours in 20% sucrose and 12 hours in 30% sucrose). The tissue was embedded in OCT (TissueTek) and 5‐µm frozen sections were obtained by cryosection. The specimens were blocked using blocking buffer solution (1× PBS/5% normal donkey serum/0.2% Triton X‐100 and 5% bovine serum albumin) for 90‐120 minutes at 4°C and then were incubated overnight with anti‐insulin (1:100; eBioscience, San Diego, CA, USA) and ant‐Ki67 (1:400; Cell Signalling Technology), anti‐glucagon (1:250; Abcam), anti‐somatostatin (1:250; Abcam). After washing three times with PBS, the slices were incubated at 4°C for 4 hours with donkey anti‐rabbit antibody (cat. # A‐31572; Alexa Fluor 555; Invitrogen) or donkey anti‐rat antibody (cat. #ab150154; Thermo Fisher). Finally, the slices were stained with 4,6‐diamidino‐2‐phenylindole (DAPI 1:1000; Invitrogen), followed by three times washing with PBS, and the slides were subjected to fluorescence microscopy analysis.35, 38

HLSC and HLSC-ILS were lysed at 4 °C for 1 h in RIPA buffer added with phosphatase and protease inhibitors (Sigma) and centrifuged at 15,000 g. After addition of lysis buffer, HLSC structures were subjected to 2 cycles of 30 s of sonication with a pause of 10 s on ice. Sonication was necessary to obtain disintegration of the capsule enveloping the structures. Aliquots containing 30 μg of proteins were quantified by Bradford method, loaded in a 4–15% gradient Tris HCl gel electrophoresis under reducing conditions and electro-blotted onto nitrocellulose membranes. The membranes were blocked with non-fat milk and overnight incubated with primary antibodies: anti-C peptide (Abcam); anti-Glucagon (Abcam) and anti-Actin (Santa Cruz Biotechnology) at the appropriate concentration. After extensive PBS-T washings, the membranes were incubated with the specific secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature, washed with PBS-T, developed with ECL detection reagents, and subjected to Chemidoc (Bio-Rad, Hercules, CA).

A-IDE-KO and control mouse pancreas sections were stained with the following antibodies diluted in blocking solution (1% BSA, 0.2% normal goat serum in PBS): anti-glucagon (Abcam, UK), anti-IDE (Millipore, USA), anti-Ki67 (Invitrogen, USA), anti-vesicle-associated membrane protein 2 (VAMP-2) (Cell Signaling, USA) and anti-α-synuclein (Santa Cruz Biotechnology, USA). Secondary antibodies were incubated in blocking solution as well. See ESM Table 2 for further details. Immunofluorescence intensity quantification of IDE, VAMP-2 and α-synuclein were performed as previously described [13 (link)].
See ESM Methods for further details.