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7 protocols using mouse monoclonal anti human glucagon

1

Pancreatic Tissue Sectioning and Immunostaining

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The pancreas was divided into consecutive tissue blocks (~5 mm) with alternating collection between paraffin-embedding and snap frozen18 (link). In this study, paraffin-embedded sections (5 μm) were stained with the following primary antibodies (all 1:500): polyclonal guinea pig anti-porcine insulin (DAKO, Carpinteria, CA), mouse monoclonal anti-human glucagon (Sigma-Aldrich, St. Louis, MO), polyclonal goat anti-somatostatin (Santa Cruz, Santa Cruz, CA), polyclonal goat anti-human PP (DAKO), and DAPI (Invitrogen, Carlsbad, CA). The primary antibodies were detected using a combination of DyLight 488, 549, and 649-conjugated secondary antibodies (1:200, Jackson ImmunoResearch Laboratory, West Grove, PA).
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2

Multimodal Immunohistochemistry Analysis

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Paraffin-embedded sections (5 μm) were stained with the following primary antibodies (all 1:500): polyclonal guinea pig anti-porcine insulin (DAKO, Carpinteria, CA), mouse monoclonal anti-human glucagon (Sigma-Aldrich, St. Louis, MO), polyclonal goat anti-somatostatin (Santa Cruz, Santa Cruz, CA), monoclonal mouse anti-human CD45 (DAKO), and DAPI (Invitrogen, Carlsbad, CA). The primary antibodies were detected using a combination of DyLight 488, 549, and 649-conjugated secondary antibodies (1:200, Jackson ImmunoResearch Laboratory, West Grove, PA).
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3

Immunostaining of Isolated Islets

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Isolated islets were stained with the following primary antibodies (all 1∶500): polyclonal guinea pig anti-porcine insulin (DAKO, Carpinteria, CA), mouse monoclonal anti-human glucagon (Sigma-Aldrich, St. Louis, MO), polyclonal goat anti-somatostatin (Santa Cruz, Santa Cruz, CA), and DAPI (Invitrogen, Carlsbad, CA). The primary antibodies were detected using a combination of DyLight 488, 549, and 649-conjugated secondary antibodies (1∶200, Jackson ImmunoResearch Laboratory, West Grove, PA).
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4

Histological Analysis of Mouse Pancreas

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Mouse pancreata were excised and frozen or paraffin-embedded. Sections were stained with hematoxylin and eosin (H&E), and immunostained with the following primary antibodies (all 1:500): rat anti-mouse CD4 and CD8 (BD Biosciences Pharmingen, San Diego, CA), polyclonal guinea pig anti-porcine insulin (DAKO, Carpinteria, CA), mouse monoclonal anti-human glucagon (Sigma-Aldrich, St. Louis, MO) and polyclonal goat anti-somatostatin (Santa Cruz, Santa Cruz, CA). Nuclei were stained with DAPI (Sigma-Aldrich). The primary antibodies were detected using ABC staining (Thermo Fisher Scientific, Rockford, IL) or a combination of DyLight 488, 549, and 649-conjugated secondary antibodies (1:200, Jackson ImmunoResearch Laboratory, West Grove, PA).
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5

Immunohistochemical Analysis of Pancreatic Tissue

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At the time of necropsy, pancreata were dissected and fixed in 4% paraformaldehyde overnight and paraffin-embedded. Tissue sections (5 μm in thickness) were immunostained with the following primary antibodies (all at 1:500 dilution): polyclonal guinea pig anti-porcine insulin (DAKO, Carpinteria, CA), mouse monoclonal anti-human glucagon (Sigma-Aldrich, St. Louis, MO), polyclonal goat anti-somatostatin (Santa Cruz, Santa Cruz, CA), and DAPI (Invitrogen, Carlsbad, CA). The primary antibodies were detected using a combination of DyLight 488, 549, and 649-conjugated secondary antibodies (1:200, Jackson Immuno Research Laboratory, West Grove, PA).
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6

Immunohistochemical Analysis of Pancreas

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At the time of tissue harvest, the pancreas was dissected, weighed, and fixed in 4% paraformaldehyde overnight followed by paraffin embedding. Pancreas sections (5 μm in thickness) were immunostained with the following primary antibodies at 1:500 dilution: polyclonal guinea pig anti-porcine insulin (DAKO, Carpinteria, CA), mouse monoclonal anti-human glucagon (Sigma-Aldrich), polyclonal goat anti-somatostatin (Santa Cruz Biotechnology, Santa Cruz, CA), and DAPI (Invitrogen, Carlsbad, CA). The primary antibodies were detected using a combination of DyLight 488-, 549-, and 649-conjugated secondary antibodies (1:200, Jackson Immuno Research Laboratory, West Grove, PA). Antibodies used in this study have been previously validated [36 ].
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7

Immunohistochemical Analysis of Pancreatic Islets

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The pancreas was divided into consecutive tissue blocks (~5 mm) with alternating collection between paraffin-embedding and snap frozen18. In this study, paraffin-embedded sections (5 μm) were stained with the following primary antibodies (all 1:500): polyclonal guinea pig anti-porcine insulin (DAKO, Carpinteria, CA), mouse monoclonal anti-human glucagon (Sigma-Aldrich, St. Louis, MO), polyclonal goat anti-somatostatin (Santa Cruz, Santa Cruz, CA), and DAPI (Invitrogen, Carlsbad, CA). The primary antibodies were detected using a combination of DyLight 488, 549 and 649-conjugated secondary antibodies (1:200, Jackson ImmunoResearch Laboratory, West Grove, PA).
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