Pancreata from mice were fixed with 10% zinc formalin overnight and paraffin embedded. 5-μm sections of the pancreata were generated, and staining was performed after blocking with 5% normal goat serum with anti-Insulin (Linco) and anti-Glucagon (Cell Signaling) antibodies using established protocols. Antigen retrieval was prepared by using citrate buffer pH 6.0. After staining, slides were mounted with antifade mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Immunofluorescent images of pancreatic sections were obtained using a Nikon Storm/Tirf/Epifluorescence. Images used for β-cell mass calculations were obtained with an EVOS FL Auto imaging system. The images of hematoxylin and eosin (H&E)-stained pancreatic sections were obtained using an AmScope light microscope. For analysis of β-cell mass and α:β cell ratio, the images were analyzed by using the Nikon Elements Advanced Research software program.
Anti glucagon
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-glucagon is a primary antibody that specifically binds to the glucagon protein. It is used in various laboratory techniques to detect and quantify glucagon levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti insulin, by Cell Signaling Technology (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Storm tirf epifluorescence, by Nikon (2 mentions)
Elements advanced research software program, by Nikon (2 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions)
Tcs sp8 sted 3x, by Leica (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Abcam (1 mentions)
Ab3623, by Merck Group (1 mentions)
Ab56416, by Abcam (1 mentions)
Anti insulin, by Merck Group (1 mentions)
Gp62 c, by Progen Biotechnik (1 mentions)
Ab168831, by Abcam (1 mentions)
L7543, by Merck Group (1 mentions)
Ta50011 100, by OriGene (1 mentions)
Alx 803 080, by Enzo Life Sciences (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Fluorescence conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti insulin, by Abcam (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Anti pdx1, by Merck Group (1 mentions)
13 protocols using anti glucagon
1
Pancreatic Immunofluorescence Histology Protocol
2
Immunofluorescent Analysis of Pancreatic Islets
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Pancreatic tissue sections were incubated with primary antibodies overnight at 4 °C. The primary antibodies were as follows: anti-Glucagon (Cell Signaling Technology, Cat#2760), anti-insulin (Cell Signaling Technology, Cat#3014). The sections were stained with secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit IgG, Abcam, Cat#ab150077) for 2 h at 4 °C in dark. After washing with PBS, the sections were stained with DAPI and observed by confocal microscopy (Leica TCS SP8 STED 3X).
3
Immunofluorescence and Immunoblot Antibody Protocols
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Primary antibodies used for confocal microscopy or immunoblot analysis were as follows: anti-insulin (I2018, Sigma-Aldrich; 18-0067, Invitrogen, Camarillo, CA, USA), anti-glucagon (8233; Cell Signaling Technology, Danvers, MA, USA), anti-active caspase-3 (AB3623; Millipore Corp.), anti-8-OHdG (MOG-020P; JaICA, Shizuoka, Japan), anti-p62 (AB56416, Abcam, Cambridge, UK; GP62-C, Progen Biotechnik GmbH, Heidelberg, Germany), anti-ubiquitin (PA5-17067; Thermo Fisher Scientific, Rockford, IL, USA), anti-LC3B (L7543, Sigma-Aldrich; AB168831, Abcam; ALX-803-080, ENZO Life Sciences, Inc., Farmingdale, NY, USA), anti-LAMP-2A (3900-100; BioVision Inc., Milpitas, CA, USA), and anti-DDK (TA50011-100, OriGene Technologies).
4
Immunofluorescent Analysis of Pancreatic Islet Cells
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On day 15 of IPCs differentiation, clusters were washed with PBS and fixed using 4% paraformaldehyde in PBS at 4°C overnight. The fixed clusters were washed with PBS, then blocked with PBS, containing 1% BSA and 0.2% Triton X-100, for 1 h at room temperature, and incubated overnight with anti-Pdx1 (Millipore, Germany), anti-insulin (Abcam, UK), anti-glucagon (Cell signaling, USA), and anti-c-peptide (R&D systems, USA) at 4°C overnight. Clusters were then stained with a fluorescence-conjugated secondary antibody (Life Technologies, Carlsbad, CA) for 2 h at room temperature, the nucleus was stained with DAPI (5 μg/ml) for 10 min, mounted with VECTASHIELD (Vector Laboratories, USA), and observed under a confocal microscope (Zeiss, Germany). To evaluate PDX-1, insulin, glucagon and c-peptide, 8~15 clusters were imaged from three independent experiments. Image J program (https://imagej.nih.gov/ij/ ) was used to quantify the fluorescence intensity.
5
Differentiation and Characterization of hESC-derived Cells
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hESC-derived cells were dissociated using Accutase. To analyze GFP expression, the cells were resuspended in PBS and used directly for analysis. For intracellular staining, the cells were fixed and stained using Foxp3 staining buffer set (eBiosciences) according to the manufacturer’s instructions. Briefly, cells were first blocked with 2% horse serum for 15 min and then incubated with primary antibody for 45 min at RT, washed twice, incubated with fluorescence-conjugated secondary antibody for 30 min at 4 °C, washed twice and re-suspended in FACS buffer for analysis. The following primary antibodies were used: anti-SOX17 (1:500, R&D), anti-PDX1 (1:500, R&D), anti-pro-insulin (1:500, Millipore), anti-glucagon (1:100, Cell Signaling), anti-somatostatin (1:1000, DAKO), and anti-ghrelin (1:500, Santa Cruz). Samples were analyzed with an Accuri C6 flow cytometry instrument and the data were processed using Flowjo v10 software.
6
Immunohistochemical Analysis of Pancreatic Specimens
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Pancreatic specimens were fixed with 10% buffered formalin, dehydrated in ethanol, embedded with paraffin, and stained with hematoxylin and eosin. Standard procedures for IHC analysis were performed according to manufacturer’s protocol. Deparaffinize and rehydrate formalin-fixed paraffin-embedded tissue section. Add enough drops of Hydrogen Peroxide Block to cover the sections. Apply Protein Block and incubate for 5 minutes at room temperature to block nonspecific background staining. Apply primary antibody and incubate according to manufacturer's protocol. Apply Biotinylated goat anti rabbit IgG(H+L) and incubate for 10 minutes at room temperature. Apply Streptavidin Peroxidase and incubate for 10 minutes at room temperature. Add 20ul DAB Chromogen to 1 mL of DAB Substrate, mix by swirling and apply to tissue. Add enough drops of Hematoxylin to cover the section. Rinse 7–8 times in tap water. Add Mounting Medium to cover the section. The image of the IHC stained slides were captured using a Carl Zeiss Axioskop 2 plus microscope (Carl Zeiss, Thornwood, NY). The following primary antibodies were used: Anti-glucagon (Cell Signaling Technology, Cat# 2760); Anti-cytokeratin 19 (GeneTex, GTX112666); Anti-insulin (Cell Signaling Technology, Cat# 3014); Anti-ARID1A (Abnova, MAB15809); Anti-IL-1β (Abcam, ab9722); Anti-F4/80 (Cell Signaling Technology, Cat# 70076).
7
Immunofluorescence Imaging of Pancreatic Islets
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Formalin-fixed pancreatic samples were embedded into paraffin blocks. Representative sections (5 µm) were incubated overnight with the following primary antibodies: anti-glucagon (1:200; Cell Signaling Technology, Danvers, MA, USA); anti-insulin (1:400; Cell Signaling Technology or Proteintech Group, Rosemont, IL, USA); anti-Neurogenin 3 (Ngn3) (1:50; Santa Cruz Biotechnology, Dallas, TX, USA); anti-synaptophysin (Syn) (1:50; Cell Signaling Technology); anti-somatostatin (Ssn) (1:50; Proteintech Group); anti-Forkhead box-containing protein O1 (Foxo1) (1:100; Cell Signaling Technology); or anti-Notch1 (1:50; Cell Signaling Technology). After incubation with ?uorescein-labeled anti-rabbit and anti-mouse secondary antibodies, the slides were mounted and imaged via confocal microscope. For quantitative analytics, we scored at least three random sections per pancreas and five random islets per section.
SAL restores morphology and architecture of islets in OLETF rats ![]()
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8
Immunofluorescence of Pancreas Samples
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Mice pancreas samples were collected then rinsed in PBS and fixed in 4% paraformaldehyde overnight before being dehydrated and embedded into paraffin. After that, samples were sectioned into 4 μm slices. The immunofluorescence staining was performed as previously described [12 (link)]. The following primary antibodies were used: anti-insulin (cat. no. C27C9, Cell Signaling Technology, Danvers, MA, USA), anti-glucagon (cat. no. ab10988, Abcam, Cambridge, UK). The nucleus was stained with Hoechst 33342 (Eugene Oregon, USA). The images were captured by a 40x objective with an LSM 510 confocal microscope (Zeiss, Jena, Germany).
9
Characterization of hESC-derived cells
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hESC-derived cells were dissociated by Accutase (STEM CELL Technologies). The resulting single cell suspension was fixed and permeabilized using FOXP3 staining buffer set (eBiosciences) for 1 h at room temperature following manufacturer’s instruction. The cells were then stained with primary antibodies at 4C overnight. Primary antibodies were anti-NKX6.1 (1:500), anti-Glucagon (1:500, cell signalling), anti-c-peptide (1:500, University of Iowa Hybridoma bank), anti-insulin (1:500), anti-somatostatin (1:500, Novus biologicals) and anti-pancreatic polypeptide (1:1000, DAKO). After washing, cells were incubated with secondary antibodies for 1 h at room temperature. Alexa 647 conjugated donkey anti-mouse or Alexa 488 conjugated donkey anti-guinea pig secondary antibody (1:500) Flow cytometry data were obtained using an Accuri C6 flow cytometry instrument and analyzed with FlowJo.
10
Pancreatic Immunofluorescence Histology Protocol
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Pancreata from mice were fixed with 10% zinc formalin overnight and paraffin embedded. 5-μm sections of the pancreata were generated, and staining was performed after blocking with 5% normal goat serum with anti-Insulin (Linco) and anti-Glucagon (Cell Signaling) antibodies using established protocols. Antigen retrieval was prepared by using citrate buffer pH 6.0. After staining, slides were mounted with antifade mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Immunofluorescent images of pancreatic sections were obtained using a Nikon Storm/Tirf/Epifluorescence. Images used for β-cell mass calculations were obtained with an EVOS FL Auto imaging system. The images of hematoxylin and eosin (H&E)-stained pancreatic sections were obtained using an AmScope light microscope. For analysis of β-cell mass and α:β cell ratio, the images were analyzed by using the Nikon Elements Advanced Research software program.
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