Islet area: 10–12 pancreatic sections (5 μm) separated by 200 μm were stained with H&E and area of individual circled islets calculated using Matlab. β- and α-cells were stained using anti-insulin (1:100, Dako) or anti-glucagon (1:50, Sigma). EM: whole islets were fixed and embedded in Epon. Thin sections (60 nm) were viewed by Tecnai 12 transmission electron microscope at 120 kV. Mitochondrial surface area was calculated from 250 single mitochondria (∼30 β-cells).
Anti glucagon
Manufactured by Merck Group
Sourced in United Kingdom, United States, Spain
Anti-glucagon is a laboratory equipment product used for the detection and quantification of glucagon, a hormone produced by the pancreas. It functions as a tool for researchers and scientists to analyze and study glucagon levels in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti insulin, by Agilent Technologies (7 mentions)
Anti insulin, by Merck Group (5 mentions)
Anti somatostatin, by Santa Cruz Biotechnology (3 mentions)
Anti somatostatin, by Agilent Technologies (2 mentions)
Anti pc1 3, by Merck Group (2 mentions)
Anti nkx6, by Novo Nordisk (2 mentions)
Anti neurod1, by Merck Group (2 mentions)
Insulin, by Agilent Technologies (1 mentions)
London resin gold, by Agar Scientific (1 mentions)
Anti fading mounting medium, by Vector Laboratories (1 mentions)
Alkaline phosphatase conjugated sheep anti dig antibody, by Roche (1 mentions)
Anti gfp, by Cell Signaling Technology (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Anti nkx2, by Santa Cruz Biotechnology (1 mentions)
Collagenase, by Merck Group (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
Anti insulin, by Abcam (1 mentions)
Anti ki67, by BD (1 mentions)
Anti phospho histone h3 phh3, by Merck Group (1 mentions)
20 protocols using anti glucagon
1
Pancreatic Islet Morphometry and Ultrastructure
2
Immunogold Labeling of Endocrine Cells
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EndoC-βH1 and -βH2 cells cultured either in flasks or on a 0.4 μm polycarbonate membrane (Nunc, #137052) were fixed in 2.5% glutaraldehyde, post-fixed in 2% uranyl acetate, dehydrated in graded methanol, and embedded in London Resin Gold (Agar Scientific, Stansted, UK). Ultrathin sections (70 nm) cut onto nickel grids were immunolabelled with anti-glucagon (Sigma, 1:100) or anti-somatostatin (Santa Cruz Biotechnology, #25262, 1:10) followed by anti-rabbit biotin (Vector Laboratories, Peterborough) and streptavidin gold 15 nm (British Biocell International, Cardiff, UK). Insulin was immunolabelled (DAKO, Ely, UK, 1:500) followed by anti-guinea pig gold 10 nm (British Biocell International). Sections were viewed on a Joel 1010 microscope (accelerating voltage 80 kV) with a digital camera (Gatan, Abingdon). The analysis is detailed in the Supllementary material.
3
Pancreatic CB1 Receptor Regulation
4
Histological Analysis of Pancreatic Tissue
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Pancreas was dissected, fixed in 4% freshly prepared paraformaldehyde (pH 7.4) for 24 h at 4 °C, and then processed routinely for paraffin embedding23 (link). For miRNA in-situ hybridization, sections were first deparaffinized and rehydrated, then treated with Proteinase K (Roch, 40 µg/ml) as described25 (link). Briefly, a total of 3 pmol of DIG-labeled Locked Nucleic Acid (LNA) probe for miR-30d (Exiqon) were mixed with 200 µl of hybridization buffer and applied onto the slides in order to hybridize at 37 °C overnight. Slides were then washed using 2X SSC (saline-sodium citrate) solution and incubated with alkaline phosphatase-conjugated sheep anti-DIG antibody (Roche) at 4 °C overnight. Alkaline phosphatase reaction was carried out with 50 mg/ml of nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) staining solution at room temperature overnight.
For immunohistochemistry, sections were immunostained with anti-insulin (Sigma), anti-glucagon (Sigma), or anti-GFP (Cell signaling) for overnight incubation at 4 °C. The immunodetection was processed with Alexa Fluor 488- or Alexa Fluor 596-conjugated secondary antibodies (Invitrogen) for 2 h at room temperature. Slides were then mounted with anti-fading mounting medium (Vector Labs) and the images were captured on Olympus FluoView FV1000 confocal microscopy or Leica fluorescence microscopy.
For immunohistochemistry, sections were immunostained with anti-insulin (Sigma), anti-glucagon (Sigma), or anti-GFP (Cell signaling) for overnight incubation at 4 °C. The immunodetection was processed with Alexa Fluor 488- or Alexa Fluor 596-conjugated secondary antibodies (Invitrogen) for 2 h at room temperature. Slides were then mounted with anti-fading mounting medium (Vector Labs) and the images were captured on Olympus FluoView FV1000 confocal microscopy or Leica fluorescence microscopy.
5
Immunohistochemical Quantification of Lipid Droplets
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EndoCβH2-Cre cells cultured on chamber slides were fixed at room temperature for 12 min with 4% paraformaldehyde-PBS, permeabilized for 8 min with 0.5% Triton-PBS, and blocked for 30 min with 0.5% BSA-PBS followed by a 4°C overnight incubation with one of the following primary antibodies: anti-insulin (guinea pig, 1:500; Dako, Santa Clara, CA), anti-glucagon (mouse, 1:400; Sigma-Aldrich), anti-somatostatin (goat, 1:400; Santa Cruz Biotechnology, Santa Cruz, CA), anti-FEV (rabbit, 1:400; Thermo Fisher Scientific), anti-MAFA (rabbit, 1:500, cat. no. NBP1-00121; Novus), and anti-NKX2.2 (anti-goat, 1:400, Santa Cruz Biotechnology). Species-matched antibodies conjugated with the Cy2, Cy3, or Cy5 fluorophores were used for secondary detection (1:1,000; Jackson ImmunoResearch, West Grove, PA). BODIPY 493/503 (5 μmol/L in PBS; Thermo Fisher Scientific) was used to detect neutral lipid enriched LDs with incubation at room temperature for 30 min following the secondary antibody treatment. Images were acquired on a ZEISS Axio Imager M2 wide-field microscope with Apotome. Quantification of the LD level was calculated as the BODIPY 493/503 area divided by the DAPI+ nuclear cell number with ImageJ software. Normalization was to the Sham control; at least five distinct areas of the slide from several independently generated sample sets were quantified per condition.
6
Whole-Mount Immunohistochemistry of Zebrafish Larvae
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Whole-mount immunohistochemistry was performed as described before [86 (link)], with incubation in 0.1% collagenase (Sigma) in PBS-Tween for 30 min for the digestion step. For immunohistochemistry against glucagon and somatostatin, larvae were deyolked before incubation in blocking buffer. Primary antibodies used were anti-cytokeratin (Santa Cruz Biotechnology, 1:50), anti-glucagon (Sigma, 1:1,000), anti-somatostatin (Dako, 1:200) and anti-carboxypeptidase-a (Sigma, 1:100). The larvae were mounted with Vectashield Mounting Media (Vector Laboratories).
7
Pancreas and Islet Cell Analysis
8
Phospho-AMPK Thr 172 Quantification
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Slides were prepared from isolated pancreata and visualized as previously detailed (11 (link)). ImageJ software was used to calculate the mean intensity on phospho-AMPK Thr 172 in the β-cell area and in the acinar tissue surrounding the islets. Western blotting was performed as previously described (11 (link)) with 100–150 human or mouse islets. Anti–acetyl-CoA-carboxylase (ACC), anti-phospho-ACC, anti-AMPK, anti–phospho-AMPK Thr 172, anti-Raptor, and anti–phospho-Raptor were from Cell Signaling Technology (Danvers, MA, USA). Anti-glucagon was from Sigma-Aldrich (St. Louis, MO, USA).
9
Whole Mount Immunostaining of Pancreatic Islets
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Protocol for whole mount immunostaining was adapted from26 (link). Primary antibodies used were anti-Insulin (Guinea Pig from DAKO 1:200), anti-GFP (Chicken from Abcam 1:1000) and anti-Glucagon (Mouse from Sigma 1:400). Secondary antibodies used were Alexa Fluor antibodies (1:300). Z-Stacks were obtained using a LSM-780 confocal microscope unless otherwise indicated in the text. For imaging of Tg(ins:Hsa.HIST1H2BJ-GFP;ins.dsRED)s960, images were acquired by setting the gain just below the threshold of signal saturation in both GFP and dsRed channels. For image analysis, the number of green only beta-cells was counted using ZEN blue software. Beta-cells which did not have an overlapping GFP and dsRED signals were counted as GFP-positive but dsRED-negative beta-cells.
10
Immunocytochemistry for Pancreatic Cell Markers
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Cells were fixed in 4% paraformaldehyde solution (Affymetrix) for 20 min, then blocked and permeabilized in PBS solution containing 5% horse serum and 0.3% Triton for 1 h at room temperature. The cells were incubated with primary antibodies overnight at 4 °C followed by 1 h incubation with fluorescence-conjugated secondary antibodies (Alexafluor, ThermoFisher Scientific) at RT. For pSMAD2/3 staining, cells were permeabilized with ice-cold methanol at −20 °C for 10 min after fixation and prior to blocking. The following primary antibodies were used: anti-OCT4 (1:200, Santa Cruz), anti-SOX17 (1:500, R&D), anti-PDX1 (1:500, R&D), anti-SOX9 (1:1000, Millipore), anti-NKX6.1 (1:500, DSHB), anti-NKX2.2 (1:500, DSHB), anti-PAX6 (1:1000, Covance), anti-ISL1 (1:200, DSHB), anti-UCN3 (1:500, Pheonix Pharmaceuticals), anti-NGN3 (1:500, R&D), anti-chromogranin A (1:1000, Immunostar), anti-glucagon (1:2000, Sigma), anti-somatostatin (1:1000, DAKO), anti-ghrelin (1:500, Santa Cruz), anti-insulin (1:500, DAKO), and anti-cleaved caspase-3 (1:1000, BD Biosciences), anti-pSMAD2/3 (1:200, Cell Signaling).
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