Following euthanasia, the pancreas of each mouse was rapidly immersion-fixed in formaldehyde, embedded in paraffin and cut in sections, rehydrated in a graded series of alcohols, washed in H2O followed by antigen retrieval (Retrieve-ALL.1, Covance) and immunostained with mouse anti-glucagon (Abcam, 1:500) and guinea pig anti- insulin (Linco, 1:1000). The appropriate secondary antibodies were conjugated to AlexaFluor- 555 or 647 (Invitrogen). Slides were mounted with Prolong Gold (Invitrogen) and imaged by epifluorescence in an Olympus FV500 confocal microscope with 60x (NA 1.4) oil objectives. The number of alpha cells (defined as glucagon-positive) or beta cells (defined as insulin- positive) were either manually counted for each islet by a single observer in a blinded fashion and the fraction of each cell type subjected to a Kruskal-Wallis test with Dunn’s corrections for multiple testing (GraphPad Prism 6.0), or was counted by ImageJ-1.50 with the assistance of either the “cell counter plug-in” (http://rsbweb.nih.gov/ij/plugins/cell-counter.html ) or with the “ITCN 1.6 plug-in” (http://rsb.info.nih.gov/ij/plugins/itcn.html ) and the ratio between the cell types analyzed by Mann-Whitney test (GraphPad Prism 6.0). Cell ratios within individual islets were quantified and expressed as mean ± SE, with p-value < 0.05 considered statistically significant.
Mouse anti glucagon
Manufactured by Abcam
Sourced in United States, United Kingdom
Mouse anti-glucagon is a primary antibody that binds specifically to the hormone glucagon. Glucagon is a 29-amino acid peptide hormone produced by the alpha cells of the pancreas and plays a key role in regulating blood glucose levels. This antibody can be used to detect and quantify glucagon in various biological samples and research applications.
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Lab products found in correlation
Guinea pig anti insulin, by Agilent Technologies (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
710 confocal microscope, by Zeiss (3 mentions)
Guinea pig anti insulin, by Merck Group (2 mentions)
Bz analyzer, by Keyence (2 mentions)
C1 confocal microscope, by Nikon (2 mentions)
Fv500 confocal microscope, by Olympus (1 mentions)
Alexa fluor 555 or 647, by Thermo Fisher Scientific (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Prism 6, by GraphPad (1 mentions)
Pressure cooker, by Biocare Medical (1 mentions)
Mouse anti brdu, by GE Healthcare (1 mentions)
Rabbit anti glucagon, by Abcam (1 mentions)
Confocal microscope, by Nikon (1 mentions)
Anti mouse cy3, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Eclipse ti series, by Nikon (1 mentions)
Nis elements software, by Nikon (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Guinea pig anti insulin, by Abcam (1 mentions)
19 protocols using mouse anti glucagon
1
Quantifying Pancreatic Islet Cell Populations
2
Immunofluorescence Analysis of Pancreatic Islets
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Paraffin sections (5 μm thick) were rehydrated, and antigen retrieval was performed using a Biocare pressure cooker and citrate (pH 6) buffer. The following primary antibodies were used: guinea pig anti-insulin (1:200; Dako), mouse anti-glucagon (1:800; Abcam), rabbit anti-glucagon (1:8000; Abcam), and mouse anti-BrdU (1:300; GE healthcare). For DNA counterstaining we used DAPI (Sigma). Secondary antibodies from Jackson ImmunoResearch were as follows: anti-guinea pig Alexa Fluor 488 (1:200), anti-mouse Cy3 (1:500), and anti-mouse Alexa Fluor 647 (1:500). Immunofluorescence images were captured using a Nikon confocal microscope. To assess β cell replication, at least 2000 β cells were counted per animal, and at least two slides at a distance of >200 µm were analyzed.
3
Evaluating Islet Graft Function in Mice
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Human islet-engrafted mice were provided drinking water containing 0.8 mg/mL of bromodeoxyuridine (BrdU) ad libitum for 7 days prior to nephrectomy of the graft-bearing kidney. Euglycemic mice at the time of nephrectomy were followed for reversion to hyperglycemia for confirmation of human islet graft function. Islet graft-bearing kidneys were fixed in 10% neutral-buffered formalin. Paraffin-embedded sections were stained with guinea pig anti-insulin (Dako, Carpinteria, CA, USA), mouse anti-glucagon (Abcam, Cambridge, England), and rat anti-BrdU (Accurate Chemical, Westbury, NJ, USA); secondary Alexa Fluor antibodies (Alexa Fluor 647, 594, 488) were from Life Technologies (Carlsbad, CA, USA), and 4′,6-diamidino-2-phenylindole (DAPI) was from Sigma-Aldrich Co. Insulin+, insulin+BrdU+, glucagon+, and glucagon+BrdU+ cells were visualized by fluorescence microscopy (Nikon Eclipse Ti series; Nikon Corporation, Tokyo, Japan). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed as per manufacturer’s instructions (Hoffman-La Roche Ltd). All counts were performed with Nikon NIS Elements software.
4
Immunofluorescence Analysis of Pancreatic Islets
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FFPE pancreas sections of 4 µm thickness were dewaxed and rehydrated according to standard protocols. The sections were immersed in a sodium citrate buffer (10 mM sodium citrate, pH 6.0) and subjected to heat-induced epitope retrieval. After rehydration in PBS, the sections were treated with a serum-free protein block (Dako, North Sydney, NSW, Australia) for 20 min, followed by primary antibody incubations at 4 °C overnight using guinea pig anti-insulin (1:200, Abcam, Melbourne, VIC, Australia), mouse anti-glucagon (1:200, Abcam, Melbourne, VIC, Australia), and rabbit anti-somatostatin (1:250, Abcam, Melbourne, VIC, Australia), all diluted in antibody diluent (Dako, North Sydney, NSW, Australia). Visualisation was performed using Alexa Fluor dyes (Life Technologies, Scoresby, VIC, Australia) and was imaged using a confocal Nikon A1+ point scanning confocal microscope.
5
Immunohistochemical Analysis of Pancreatic Tissue
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Adult mouse pancreatic tissues were harvested and processed, as previously described18 . After antigen retrieval on tissue sections, overnight incubation was carried out with each of the primary antibodies: guinea pig anti‐insulin (1:4,000; Sigma, St. Louis, MD, USA), mouse anti‐glucagon (1:4,000; Abcam, Cambridge, MA, USA) and rabbit anti‐ADM (1:200; Santa Cruz Biotechnology). Coverslips were placed in antifade solution containing 4′,6‐diamidino‐2‐phenylindole (Vectashield; Vecta Laboratories, Burlingame, CA, USA). To visualize the antigens, fluorescein FITC and cyanine Cy3‐conjugated appropriate secondary antibodies (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used. Digital images acquired were compiled using a BZ Analyzer (Keyence, Osaka, Japan).
6
Immunofluorescent Staining of Paraffin Embedded Tissue
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Immunofluorescent staining was performed as previously published [22 (link)] with modifications to account for the use of paraffin embedded tissue. Briefly, tissue sections were deparaffinized through graded ethanols (100%, 95%, 85%, 75%, 50%; Fisher Scientific) and then blocked using normal donkey serum (Sigma). Primary antibodies used included guinea pig anti-insulin (DAKO; 1:500), mouse anti-glucagon (Abcam; 1:500), rat anti-somatostatin (abcam; 1:200), goat anti-pancreatic polypeptide (abcam; 1:200), goat anti-ghrelin (Santa Cruz; 1:500), mouse anti-Pax5 (DAKO; 1:200), mouse anti-CD8 (Thermo Fisher; 1:500), mouse anti-CD4 (Leica; 1:500), rabbit anti-eIF5AHyp ([13 (link),18 (link)]; 1:1000). Secondary antibodies including Alexa-488, Cy3, or Alexa-647 (Jackson Immunoresearch) were used to visualize primary antibodies. DAPI (Sigma; 1:1000) was used to visualize nuclei. Images were acquired with a Zeiss 710 confocal microscope.
7
Immunohistochemical Analysis of Pancreatic Islets
8
Immunofluorescent Characterization of 3D Liver and Pancreatic Tissues
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Liver spheroids and pancreatic islet microtissues were embedded in Tissue-Tek® O.C.T. compound (Sakura Finetek, Torrance, CA, USA) for immunohistochemistry. Central cryostat sections of 12 µm were fixed in acetone at −20 °C for 10 min, washed with PBS and blocked with 10% (v/v) goat serum in PBS for 20 min. The pancreatic islet microtissues were immunostained with guinea pig anti-insulin antibody (Dako, Glostrup, Denmark) and mouse anti-glucagon (Abcam, Cambridge, UK) overnight, washed with PBS and, subsequently, developed by goat anti-guinea pig CF594 (Biotium, Fremont, CA, USA) and goat anti-mouse Alexa-488 (Life Technologies, Carlsbad, Ca, USA) for 45 min. DAPI was added for nuclei staining. The same procedure was carried out for the liver spheroids using mouse anti-cytokeratin 8/18 (Santa Cruz, Heidelberg, Germany), rabbit anti-vimentin (Santa Cruz), goat anti-albumin FITC (Bethyl Laboratories, Montgomery, Tx, USA) and mouse anti-Cyp3A4 (Santa Cruz) as primary antibodies and goat anti-mouse Alexa-488 (Life Technologies, Carlsbad, Ca, USA), goat anti-rabbit CF594 (Biotium) or goat anti-mouse CF594 (Biotium) as secondary antibodies. Images were obtained using a Keyence fluorescence microscope
9
Ultrastructural Analysis of Insulin and Glucagon
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To analyze the granular ultrastructure, the cells were fixed with 1% glutaraldehyde and 1% PFA in 0.1 M sodium cacodylate buffer (pH 7.2) at 4 °C. Specimens were then fixed in 2% osmium tetroxide for 60 min at 4 °C. Dehydration of the fixed samples was performed, and the samples were transferred to Lowicryl resin (Polyscience, Niles, IL, USA). Samples were then sectioned (60 nm) with an ultramicrotome (UltracutUCT, Leica, Wetzlar, Germany) and collected on nickel grids. Post-embedding immunogold labeling was performed for insulin and glucagon labeling using the rabbit anti-insulin (Abcam), mouse anti-glucagon (Abcam), 5-nm colloidal gold conjugated to goat anti-rabbit IgG (Sigma-Aldrich), and 9–11-nm colloidal gold conjugated to goat anti-mouse IgG (Sigma-Aldrich). Following immunogold labeling, the sections were double-stained with 2% uranyl acetate for 20 min and lead citrate for 10 min. The sections were then viewed using TEM.
10
Histological Analysis of Transplanted Implants
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The implants were removed, fixed in 4% formaldehyde, embedded in paraffin, and stained as described in [8 (link)], using anti-human HLA-A (abcam ab52922, 1:100), anti-insulin (Sigma, clone K36 AC10, 1:75), and anti-human CD31 (Dako Cytomation, Clone JC70A, M0823, 1:50 dilution) antibodies. Analysis of HLA-positive cells was done by counting cells in random five × 40 fields from each implant. Histological analysis of microvessel density (MVD) was performed as described previously [38 (link)]; microvessel density was assessed by counting CD31 positive red blood cell–filled lumens. Values reported for each experimental condition correspond to the average MVD value ± standard error of the mean (SEM) obtained from all the individual implants.
Immunofluorescence: antibodies used in this study included guinea pig anti-insulin (1:400; Dako) and mouse anti-glucagon (1:800; Abcam). Fluorescent images were taken on a Nikon C1 confocal microscope at an original magnification × 40 as described [45 (link)].
Immunofluorescence: antibodies used in this study included guinea pig anti-insulin (1:400; Dako) and mouse anti-glucagon (1:800; Abcam). Fluorescent images were taken on a Nikon C1 confocal microscope at an original magnification × 40 as described [45 (link)].
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