Ab7842

Manufactured by Abcam

Sourced in United States, United Kingdom, Japan

Ab7842 is a laboratory equipment product. It is a device used for a specific function in a laboratory setting. The core function of this product is to perform a particular task required in scientific research or analysis. No further details about the intended use or application of this product are provided.

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Lab products found in correlation

Ab10988, by Abcam (12 mentions) Lsm 710 confocal microscope, by Zeiss (6 mentions) Paraformaldehyde (pfa), by Merck Group (5 mentions) Hoechst 33342, by Thermo Fisher Scientific (5 mentions) Ab28364, by Abcam (4 mentions) A11073, by Thermo Fisher Scientific (4 mentions) Bz 9000 fluorescent microscope system, by Keyence (3 mentions) Ab6326, by Abcam (3 mentions) Alexa fluor 546, by Thermo Fisher Scientific (3 mentions) Ab218532, by Abcam (2 mentions) Ab15580, by Santa Cruz Biotechnology (2 mentions) Linco 4011, by Merck Group (2 mentions) K79bb10, by Abcam (2 mentions) Sc 56, by Santa Cruz Biotechnology (2 mentions) Sc 1506, by Cell Signaling Technology (2 mentions) Sc 260, by Santa Cruz Biotechnology (2 mentions) Sc 593, by Santa Cruz Biotechnology (2 mentions) Histostain kit, by Thermo Fisher Scientific (2 mentions) Dab substrate, by Thermo Fisher Scientific (2 mentions) A1 confocal microscope, by Nikon (2 mentions)

72 protocols using ab7842

Immunostaining and Clearing of Pancreatic Tissue

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Thick 100 μm cryostat sections were rehydrated in PBS. Sections and whole mount pancreata were blocked overnight in PBS, 2% donkey serum and 0.5% Triton-100X. The samples were incubated in primary antibodies (Chromogranin A, 1:180 from Abcam, ab15160; insulin, 1:100, from Abcam, ab7842; glucagon 1:200, from Abcam, ab10988); and Dolichos biflorus agglutinin (DBA) biotinylated (1:270, from Vectorlabs, B-1035) for 3 days at 4 °C. Secondary antibodies were applied (from Thermo Fisher Scientific) and AF647-Streptavidin (1:800, from Thermo Fisher Scientific, S21374), BV510-Streptavidin (1:600, from Biolegend, 405233) for 2 days at 4°C. All tissues were cleared with RapiClear 1.52 (from SunJin Lab, RC152001).
Thin sections were incubated in 2% Serum, 0.1% Triton-100X in PBS for 30 min, subsequently incubated in primary antibody in 0.01% Triton-100X in PBS overnight (insulin 1:100, from Abcam, ab7842, glucagon 1:200 from Abcam, ab10988, cleaved caspase 3, 1:400, from Cell Signalling, 9664S), washed 3 times for 10 min the next day, and incubated with secondary antibody in 0.01% Triton-100X for 3 h, washed and mounted.

Sznurkowska M.K., Hannezo E., Azzarelli R., Chatzeli L., Ikeda T., Yoshida S., Philpott A, & Simons B.D. (2020). Tracing the cellular basis of islet specification in mouse pancreas. Nature Communications, 11, 5037.

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Quantification of Pancreatic Islet Cell Types

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Immunohistochemistry was carried out as described previously¹⁸. Primary antibodies were: guinea pig polyclonal anti‐insulin (ab7842; Abcam, Tokyo, Japan; 1:100), mouse monoclonal anti‐glucagon (G2654, Dako Japan, Tokyo, Japan; 1:2,000), rabbit monoclonal anti‐Glp1r (ab218532; Abcam; 1:500), rabbit polyclonal anti‐Pdx1 (5796; Cell Signaling Technology, Tokyo, Japan; 1:400), rabbit monoclonal anti‐Myc (10828‐1‐AP; Proteintech Japan, Tokyo, Japan; 1:50) and rabbit monoclonal anti‐Ki67 (9129; Cell Signaling Technology, Tokyo, Japan; 1:400). The β‐cell area was detected by insulin staining, and the number of β‐cells was detected by 4′,6‐diamidino‐2‐phenylindole staining. The size of the β‐cells in each islet was calculated by the formula “total β‐cell area divided by number of β‐cells” (50 islets of 3–4 rats for each genotype). The α‐cell area was detected by glucagon staining, and ratio of the α‐ or β‐cell area in the whole pancreas was calculated by the formula “total α‐ or β‐cell area divided by whole pancreas area” (6–9 pancreas sections of 3–4 rats for each genotype).

Hayami T., Yokoi N., Yamaguchi T., Honda K., Murao N., Takahashi H., Wang S., Seino Y., Kamiya H., Yabe D., Sweet I.R., Mizoguchi A., Nakamura J, & Seino S. (2020). Tumor‐like features of gene expression and metabolic profiles in enlarged pancreatic islets are associated with impaired incretin‐induced insulin secretion in obese diabetes: A study of Zucker fatty diabetes mellitus rat. Journal of Diabetes Investigation, 11(6), 1434-1447.

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Histological Analysis of Pancreatic Tissue

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Pancreas was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned by Histology Core Facility at Cornell and University of Michigan. For Hematoxylin/eosin staining, ten-micrometer-thick paraffin sections were stained and imaged using Aperio Scanscope. For immunostaining, paraffin-embedded sections were rehydrated with sequential wash in xylene, 100%, 95%, 75% ethanol and water and boiled in 1 mM EDTA for antigen retrieval. Subsequently, sections were blocked using 5% donkey serum and incubated at 4 °C overnight with primary antibodies: Anti-insulin (Abcam ab7842 or Linco 4011, 1:200), glucagon (Sigma K79bB10, 1:1000), Ki-67 (Abcam ab15580, 1:50), Pcna (Santa Cruz sc-56, 1:100), CD31 (Santa Cruz sc-1506, 1:100), Pdx1 (Cell Signaling D59H3, 1:100), Cdk4 (Santa Cruz sc-260, 1:100) and Ccnd2 (Santa Cruz sc-593, 1:100). Following day, slides were washed and incubated with conjugated secondary antibodies and mounted on slides with prolong gold antifade/ DAPI for nuclear staining. Fluorescence Images were captured under a Nikon A1 confocal microscope at Brehm Diabetes Research Center Imaging Facility at University of Michigan. For horseradish peroxidase enzyme (HRP) staining, slides were stained with Histostain kit and DAB substrate from Invitrogen.

Ji Y., Sun S., Shrestha N., Darragh L.B., Shirakawa J., Xing Y., He Y., Carboneau B.A., Kim H., An D., Ma M., Oberhozler J., Soleimanpour S.A., Gannon M., Liu C., Naji A., Kulkarni R.N., Wang Y., Kersten S, & Qi L. (2019). Toll-like receptors TLR2 and TLR4 block the replication of pancreatic β cells in diet-induced obesity. Nature immunology, 20(6), 677-686.

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Immunocytochemical Characterization of Pancreatic Cells

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Cells were fixed with 4% PFA in PBS for ~5 min at room temperature. After blocking with 20% AquaBlock (#PP82; East Coast Bio, North Berwick, ME, USA, https://eastcoastbio.com/) for 30 min at room temperature, cells were incubated overnight at 4°C with goat anti-Pdx1 antibody (1:100; #ab47383; Abcam, Tokyo, Japan, http://www.abcam.com/), goat anti-insulin antibody (1:100; #ab7842; Abcam), or rabbit anti-C-peptide antibody (1:100; #4593; Cell Signaling, Tokyo, Japan, http://www.cellsignal.com/). This was followed by incubation for 1 h at room temperature with Alexa Fluor 488- conjugated donkey anti-goat IgG H&L (1:200; #ab150129; Abcam), FITC-conjugated anti-goat IgG (1:250; #ab6904; Abcam), or Alexa Fluor 647-conjugated anti-rabbit IgG (1:250; #4414; Cell Signaling), respectively. A medium containing DAPI (#H-1200; Vector Laboratories, Burlingame, CA, USA, https://www.vectorlabs.com/default.aspx) was used for mounting and nuclear staining.

Saitoh I., Sato M., Soda M., Inada E., Iwase Y., Murakami T., Ohshima H., Hayasaki H, & Noguchi H. (2016). Tissue-Specific Stem Cells Obtained by Reprogramming of Non-Obese Diabetic (NOD) Mouse-Derived Pancreatic Cells Confer Insulin Production in Response to Glucose. PLoS ONE, 11(9), e0163580.

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Insulin and Endothelial Cell Immunofluorescence

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All tissues were fixed with 10% buffered formalin for at least 3 days prior to processing. Samples were paraffin embedded and 5-μm sections were processed for histological analysis. Sections were deparaffinized and rehydrated prior to heat-mediated antigen retrieval (Citrate buffer, pH 6). Sections were incubated with Guinea pig anti–human insulin/glucagon antibody (AB7842; Abcam, Cambridge, UK) overnight as a primary antibody alone at 2 μg/mL at 4°C. Insulin staining was visualized with secondary goat anti–guinea pig conjugated to Rhodamine fluorochrome (Jackson ImmunoResearch, West Grove, PA) diluted 1:200 and incubated at room temperature in the dark for 1 h. Otherwise, sections were costained with cross-reactive rabbit anti–mouse CD31 as a primary antibody overnight at 4°C (1:50 dilution of Ab28364; Abcam). A horseradish peroxidase–conjugated goat anti-rabbit polyclonal (1:2,000) was used as the secondary antibody before undergoing signal amplification with Tyramide Signal Amplification Kit per the manufacturer’s instructions (Molecular Probes, ThermoFisher).

Rojas-Canales D., Walters S.N., Penko D., Cultrone D., Bailey J., Chtanova T., Nitschke J., Johnston J., Kireta S., Loudovaris T., Kay T.W., Kuchel T.R., Hawthorne W., O’Connell P.J., Korbutt G., Greenwood J.E., Grey S.T., Drogemuller C.J, & Coates P.T. (2023). Intracutaneous Transplantation of Islets Within a Biodegradable Temporizing Matrix as an Alternative Site for Islet Transplantation. Diabetes, 72(6), 758-768.

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Multiparametric Islet Immunophenotyping

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Immunohistochemistry of frozen or paraffin-embedded sections of pancreas, human islets, and human islet-like organoids (HILOs) was performed with antibodies to insulin (1/100, Abcam ab7842), c-peptide (1/100, Abcam ab30477), glucagon (1/100, Abcam ab10988), somatostatin (1/100, Abcam ab103790), pancreatic polypeptyde (1/100, Abcam, ab113694), NKX2–2 (1/100, DSHB, 74.5A5), NKX6–1 (1/100, DSHB, F55A12), MAFA (1/100, Abcam, ab26405), MAFB (1/100, Abcam, ab66506), PDX-1 (1/100, R&D, AF2419), CHGA (1/100, Abcam, ab15160), SYNAPTOPHYSIN (1/100, Biogenex, MU363-UC) and PD-L1 (1/100, Abcam, ab20592). For MAFA and MAFB, signal amplification was performed using TSA-Cy3 kit (Akoya Biosciences, SAT704A001EA). Secondary antibodies were coupled to Alexa 568, 647 (Life Technologies) and visualized by confocal microscopy (ZEISS) or fluorescence microscopy. Hoechst 33342 (Thermo Scientific, 62249, 1μg/ml final concentration) was used for nuclear staining.

Yoshihara E., O’Connor C., Gasser E., Wei Z., Oh T.G., Tseng T.W., Wang D., Cayabyab F., Dai Y., Yu R.T., Liddle C., Atkins A.R., Downes M, & Evans R.M. (2020). Immune evasive human islet-like organoids ameliorate diabetes. Nature, 586(7830), 606-611.

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Embryonic Nitrotyrosine Immunohistochemistry

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Cryostat sections (10 μm) of embryos were processed for anti-nitrotyrosine immunohistochemistry using a specific antibody (Millipore, Billerica, MA, USA; Ref. 06-284, 1:1000 dilution). Immunodetection was carried out with a secondary biotinylated goat anti-rabbit antiserum (Bio-Rad) using immunoperoxidase staining. The expression of nitrotyrosine was scored by quantifying the number of positive cells per region of interest from digital images using NIH ImageJ software. In all sections regions of interest located both on the left and right sides of the embryo were scored independently. Counterstaining was carried out with haematoxylin. Immunofluorescence on pancreas sections with an insulin antibody (Ab7842, Abcam; 1/100 dilution) was performed as described^{35 (link)}.

García-Sanz P., Mirasierra M., Moratalla R, & Vallejo M. (2017). Embryonic defence mechanisms against glucose-dependent oxidative stress require enhanced expression of Alx3 to prevent malformations during diabetic pregnancy. Scientific Reports, 7, 389.

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Quantification of Pancreatic Insulin and Glucagon

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Insulin and glucagon protein abundance was assessed using paraffin-embedded pancreatic sections from WT, PdxCreMpc2−/−, and RipCreMpc2−/− mice. Slides were rehydrated, permeabilized with 1 mg/ml trypsin, blocked in 3% BSA, and probed with guinea pig anti-insulin (Abcam #ab10988, 1:100 dilution) and mouse anti-glucagon (Abcam #ab7842, 1:100 dilution) antibodies, in 3% BSA overnight. Slides were washed and probed with Alexa Fluor^® 488 goat anti-guinea pig (Invitrogen #A11073, 1:1000 dilution) and Alexa Fluor^® 594 goat anti-mouse secondary (Invitrogen #A21125, 1:1000 dilution) antibodies in 3% BSA for 1 h. Coverslips were fixed with ProLong^® Gold antifade reagent with DAPI (Life Technologies). Stained pancreatic sections were then imaged on an EVOS FL digital inverted fluorescence microscope (Invitrogen). Islet number and cross-sectional areas were determined using ImageJ.

McCommis K.S., Hodges W.T., Bricker D.K., Wisidagama D.R., Compan V., Remedi M.S., Thummel C.S, & Finck B.N. (2016). An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion. Molecular Metabolism, 5(8), 602-614.

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Quantifying Insulin and Glucagon Levels

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The following kits were used to measure serum factors: Mouse Insulin ELISA kit (Cat # 10–1247-01) and Glucagon ELISA kit (Cat # 10–1271-01) from Mercodia (Uppsala, Sweden). Liver total acyl glycerol measurements have been described in detail in a previous study [18 (link)]. Formalin-fixed paraffin embedded pancreatic tissue was sectioned and stained as described previously [12 (link)]. Ki-67 (IHC-00375-T; Bethyl labs) and insulin (ab7842; Abcam) co-localization were imaged via a Hamamatsu NanoZoomer fluorescent slide scanner (Hamamatsu Photonics, K.K., Japan).

Burke S.J., Batdorf H.M., Huang T.Y., Jackson J.W., Jones K.A., Martin T.M., Rohli K.E., Karlstad M.D., Sparer T.E., Burk D.H., Campagna S.R., Noland R.C., Soto P.L, & Collier J.J. (2019). One week of continuous corticosterone exposure impairs hepatic metabolic flexibility, promotes islet β-cell proliferation, and reduces physical activity in male C57BL/6J mice. The Journal of steroid biochemistry and molecular biology, 195, 105468.

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Immunohistochemical Insulin Detection in Pancreas

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Animals were anesthetized and perfused intracardially with 4% paraformaldehyde/PBS. Pancreatic tissues were dissected and soaked in 4% paraformaldehyde. Fixed tissues were then processed for paraffin embedding with tissue processing. Pancreatic sections stained with Hematoxylin/Eosin. Immunohistochemical detection of insulin was performed on 4 μm sections using a standard chain polymer-conjugated technique as described previously on paraffin-embedded tissues of pancreas specimens [25 (link)]. Briefly, after deparaffinization, tissue sections were immersed in methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase activity. Protein blocker was then used for blocking non-specific binding. Antigen was retrieved by autoclaving in citrate buffer (pH 6.0). The sections were incubated with primary Anti- insulin antibody (guinea pig polyclonal to insulin, ab7842, Abcam, Cambridge, UK) at 4°C overnight and then incubated with rabbit polyclonal secondary antibody (Rabbit polyclonal secondary antibody to guinea pig IgG - H&L (HRP), ab6771, Abcam, Cambridge, UK) for 1 hour at room temperature. The slides were washed and visualized using 3, 3-diaminobenzidine, (DAB; DAKO, Glostrup, Denmark). The sections were counterstained with heamtoxylin (Dako, Glostrup, Denmark). Finally dehydration process was performed and slides were observed under light microscope.

Aali E., Mirzamohammadi S., Ghaznavi H., Madjd Z., Larijani B., Rayegan S, & Sharifi A.M. (2014). A comparative study of mesenchymal stem cell transplantation with its paracrine effect on control of hyperglycemia in type 1 diabetic rats. Journal of Diabetes and Metabolic Disorders, 13, 76.

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