Pancreata of OB and NZO mice (−CH and +CH groups, n = 3) were prepared for immunohistochemistry as previously described (7 (link)). Pancreatic sections were stained for somatostatin (1:1,000, MA5-16987; Invitrogen), insulin (1:50,000, I2018; Sigma-Aldrich), and glucagon (1:2,000, A0565; Dako). Primary antibodies were detected with fluorophore-labeled secondary antibodies at a dilution of 1:200 (anti-rat Alexa Fluor 546, anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 647; Invitrogen) for 1 h at room temperature. Images of 10–15 randomly selected islets per section were documented with a confocal microscope (TCS SP8 X; Leica Microsystems) and analyzed with ImageJ (19 (link)). In total, >6,000 islet cells were evaluated per group.
I2018
Manufactured by Merck Group
Sourced in Spain, United States
The I2018 is a laboratory instrument designed for precise and efficient sample analysis. It is a highly versatile and reliable tool that can be utilized in a variety of research and testing applications. The core function of the I2018 is to perform accurate measurements and data collection, enabling researchers and analysts to obtain reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Anti insulin, by Merck Group (2 mentions)
G2654, by Merck Group (2 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Tcs sp8 x, by Leica (1 mentions)
Ma5 16987, by Thermo Fisher Scientific (1 mentions)
A0565, by Agilent Technologies (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
D9564, by Merck Group (1 mentions)
Ab15580, by Abcam (1 mentions)
Lsm 700 meta confocal microscope, by Zeiss (1 mentions)
Alexa fluor 555 conjugated igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Anti glucagon, by Merck Group (1 mentions)
Sc 20175, by Santa Cruz Biotechnology (1 mentions)
Tcs sp2 aobs spectral confocal microscope, by Leica (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Avidin biotin complex, by Vector Laboratories (1 mentions)
Ab31419, by Abcam (1 mentions)
7 protocols using i2018
1
Immunohistochemical Analysis of Murine Pancreata
2
Immunofluorescence analysis of pancreatic islet cells
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Isolated islets were seeded in 24‐well plates and maintained in RPMI containing 10% FCS, 20 mM L‐glutamine, and 100 U/L penicillin/streptomycin with or without KY19334. For transient transfection, islets were transfected with siRNA using lipofectamine (Invitrogen) in Opti‐MEM (Gibco). Islet cells were fixed with 4% paraformaldehyde in PBS for 30 min at 20°C. The cells were then permeabilized and blocked with 0.5% Triton X‐100 and 5% BSA in PBS for 30 min at 20°C. Cells were then incubated overnight at 4°C with the following dilutions of primary antibodies: anti‐insulin (1:500, I2018, Sigma‐Aldrich) and anti‐Ki67 (1:100, ab15580, Abcam). Additionally, the cells were incubated with Alexa Fluor 488‐conjugated (1:300, A‐11001, Invitrogen) or Alexa Fluor 555‐conjugated IgG secondary antibody (1:300, A‐21428, Invitrogen) at 20°C for 1 h and counterstained with DAPI (1:5,000, D9564, Sigma‐Aldrich). The stained cells were captured using an LSM700 META confocal microscope (Carl Zeiss) after excitation with 405, 488 or 543 nm laser lines.
3
Immunohistochemical Analysis of Pancreatic Cell Types
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Pancreases from each group were excised and processed as described previously [27] (link). Sections were cut at a thickness of 5 µm, dewaxed in xylene, and rehydrated in a graded ethanol series. The sections were incubated in PBS containing 3% BSA for 1 h at room temperature to block non-specific binding and then with the following antibodies in PBS containing 3% BSA overnight at 4°C: anti-glucagon receptor (1∶100, bs-3945R; Bioss, Woburn, MA), anti-insulin (1∶100, I2018; Sigma, Madrid, Spain), anti-glucagon (1∶100, G2654; Sigma, Madrid, Spain), and anti 11b-HSD1 (1∶50, sc-20175; Santa Cruz Biotechnology). After washing, the sections were incubated with the corresponding secondary antibodies (1∶1000, Alexa Fluor; Molecular Probes, Leiden, The Netherlands) for 1 h at room temperature. Hoechst 33342 (Life Technologies, Madrid, Spain) was used for nuclear staining. Images were captured using a Leica TCS SP2 AOBS spectral confocal microscope.
4
Insulin Immunohistochemistry in Pancreatic Tissue
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Insulin-positive cells in the pancreas were detected via immunohistochemistry according to Pilz et al. [18] (link). Briefly, after dewaxing, heat-induced antigen retrieval in citrate buffer (pH 6.0) for 15 min at sub boiling temperature, and endogenous peroxidase block by 1% H 2 O 2 , a mouse anti-insulin monoclonal antibody was used as the primary antibody (#I2018, Sigma; 1:3000) for overnight incubation at 4 °C, followed by biotinylated goat anti-mouse IgG (H+L) secondary antibody (#115-065-146, Jackson ImmunoResearch Laboratories, Inc.; 1:250 + 3% porcine serum) for 1 h at room temperature, and avidin-biotin complex (#PK-6100, Vector Laboratories) for 30 min at room temperature. A horseradish peroxidase DAB substrate kit (#SK-4100, Vector Laboratories) was used to detect bound antibodies. Nuclear counterstaining was performed with Meyer's hemalum solution.
5
Immunostaining of Pancreatic Cell Markers
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Cells were cultured on glass coverslips and fixed in 2% paraformaldehyde (PFA) for 15 min. The immunofluorescence protocol performed conformed to indications provided by the supplier. The following primary antibodies were used: mouse anti-β-catenin (1/500, ab22656, Abcam), rabbit anti-BMP4 (1/500, ab39973, Abcam), rabbit anti-ROR2 (1/50, ab92379, Abcam), rabbit anti-c-JUN (1/500, ab31419, Abcam), mouse anti-insulin (1/500, I2018, Sigma-Aldrich), guinea-pig anti-porcine insulin (1/500, A056401-2, Dako), mouse anti-porcine glucagon (1/500, G2654, Sigma-Aldrich), rat anti-somatostatin (1/100, sc-47706, Santa Cruz), guinea-pig anti-PDX1 (1/500, ab47308, Abcam), and rabbit anti-NKX6.1 (1/100, NBP1-82553, Novus), rabbit anti-MAFA (1/200, ab98859, Abcam). The following secondary antibodies were used: donkey anti-rabbit A488, donkey anti-rabbit A546, goat anti-mouse A568, chicken anti-rat A647, and goat anti-guinea-pig A647 (1/500, Molecular Probes). The nuclei were stained with DAPI (D1306, Molecular Probes). The samples were mounted in Prolong Diamond Antifade Mountant Media (P36970, Life technologies) and were analyzed with Leica TCS SP5 STED CW confocal microscope. No specific feature of the original data was obscured, eliminated or misrepresented.
6
Immunofluorescent Analysis of Pancreatic Islets
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Paraffin-embedded sections (5-µm-thick) of the pancreatic splenic lobe were subjected to fluorescence immunohistochemistry as described previously (28 (link)) using a 1:200 dilution of mouse monoclonal anti-insulin antibody (I2018, Sigma Aldrich, St. Louis, MO, USA) or rabbit polyclonal antibodies against the proliferation marker, Ki67 (ab66155, Abcam, UK), and a 1:500 dilution of Alexa Flour 488 anti-mouse or Alexa Flour 555 anti-rabbit secondary antibodies (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Sections were cover-slipped with Vectashield mounting medium containing DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) and observed under fluorescence microscopy (Microscope BX60F5, Olympus) using the 40X objective, and the fluorescence signal was quantified from at least 15 islets per animal using the Image-Pro Plus 5.1 software.
7
Comprehensive Histological and Immunochemical Evaluation of Pancreatic Tissue
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