We retrieved the Matrigel, fixed in formalin, and embedded in paraffin to prepare sections of 4 µm. The sections were then deparaffinized by heating in a dry oven for 1 h and washing vigorously in xylene. Rehydration was performed serially in 100%, 90%, 80%, and 70% alcohol. The antigens were retrieved by heating the slides in 10 mM citrate buffer of pH 6.0 using a microwave (5 min, 700 w). The citric acid was neutralized by immersing the slides in 3% hydrogen peroxide for 15 min. Slides were washed in PBS and incubated overnight at 4 °C with rabbit polyclonal anti-insulin (Abcam, Cambridge, MA), anti-CD3 (Abcam), and anti-CD68 (Abcam) in a humidified chamber. The slides were incubated at room temperature for 1 h. Then, the tissue sections were incubated with peroxidase-labeled secondary antibodies (Dako, Troy, MI). The slides were counterstained with hematoxylin and eosin, gradually dehydrated using 70%, 80%, 90%, and 100% alcohol, and fixed with a coverslip using mounting medium.
Anti insulin
Manufactured by Abcam
Sourced in United Kingdom, Germany
Anti-insulin is a laboratory reagent used in research applications. It is an antibody that specifically binds to insulin, allowing for the detection and quantification of this important biomolecule. This product can be used in various analytical techniques, such as immunoassays, to study insulin levels and related biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti glucagon, by Abcam (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti il 1β, by Abcam (2 mentions)
Fluoview fv10i confocal microscope, by Olympus (2 mentions)
Cy5 or fitc coupled secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (2 mentions)
C peptide, by Developmental Studies Hybridoma Bank (2 mentions)
Rhodamine 6g, by Merck Group (2 mentions)
Rompun, by Bayer (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Fluorescein isothiocyanate fitc dextran 150 000, by Merck Group (2 mentions)
Anti cd31 dia310, by Dianova (2 mentions)
Anti ki67, by BD (2 mentions)
Anti brdu, by Agilent Technologies (2 mentions)
Dab substrate kit, by Vector Laboratories (2 mentions)
Histomouse sp kit, by Thermo Fisher Scientific (2 mentions)
Anti f4 80 antibodies, by Merck Group (2 mentions)
Anti ki67, by Abcam (2 mentions)
Peroxidase labeled secondary antibody, by Agilent Technologies (1 mentions)
28 protocols using anti insulin
1
Immunohistochemical Analysis of Pancreatic Islets
2
Islet Inflammatory and Dedifferentiation Analysis
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Islets were harvested, fixed in 4% paraformaldehyde, and immersed in 30% sucrose solution to be embedded in a frozen block. Inflammatory markers and dedifferentiation markers were detected in cell sections by immunofluorescence. Anti-FoxO1 (Santa Cruz, Dallas, Texas, USA), anti-cleaved caspase 3 (Cell Signaling, Danvers, MA, USA), anti-Oct4 (Abcam, Cambridge, UK), anti-NGN3 (Abcam), anti-A11 oligomer (Invitrogen), anti-Pdx1 (Abcam), anti-IL-1β (Abcam), and anti-insulin (Abcam) were used as primary antibodies. M1/M2 macrophage markers were identified using the antibodies of M2 macrophage differentiation markers (anti-arginase-1; Cell Signaling) and M1 macrophage differentiation markers (CD80; Abcam). Sections were incubated with primary antibody diluted in blocking buffer overnight at 4°C. Secondary antibodies (Alexa Fluor® 488-conjugated goat anti-rabbit, Alexa Fluor® 488-conjugated goat anti-mouse, and Alexa Fluor® 594-conjugated goat anti-rabbit; Abcam) were added and incubated for 1 h at room temperature. Counterstaining was performed using DAPI (1 : 10,000). Images were obtained from each section using a fluorescent microscope (Olympus, Shinjuku, Tokyo, Japan).
3
Immunohistochemical Analysis of Mitochondrial Dynamics
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For immunohistochemistry analyses cells were seeded on x-well Tissue Culture Chambers (Sarstedt, Nümbrecht, Germany) and transfected for 48 h. Cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.2% Tween20 for 5 min in phosphate-buffered saline. Cells were stained for 1 h with the primary antibodies: anti-MiD51 (1:100) (Proteintech), anti-Tom20 (1:100) (Abcam, Cambridge, UK), anti-insulin (1:100) (Abcam) and anti-LC3 (1:100) (Sigma). Cy5 or FITC-coupled secondary antibodies (1:250) were used for visualization (Molecular Probes Invitrogen, Darmstadt, Germany). Cells were mounted and counterstained using Roti®-Mount FluorCare DAPI (Roth, Karlsruhe, Germany) and analyzed using a Fluoview FV10i confocal microscope (Olympus, Hamburg, Germany).
4
Multimodal Immunofluorescence of Pancreatic Hormones
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Parafin embedded sectioned samples were stained for the following: human insulin (Anti-insulin cat# ab7842, abcam), human C-peptide (C-peptide cat #GN-1D4, Developmental Studies Hybridoma Bank, University of Iowa), NKX 6.1 (cat # F55A12, Developmental Studies Hybridoma Bank, University of Iowa), human glucagon (Anti-Glucagon cat# ab82270, abcam). Cellular nuclei were stained with DAPI (Life Technologies).
Parafin slides were deparaffinized through subsequent incubations in the following solvents (Xylene 5min 2× 100% ETOH 2min ×2 95% 2 min ×2 70% 2min ×2 d-water). Antigen retrieval was done by incubating sections for 30min in ice cooled PBS, and then blocking with 3% horse serum to block for 30 min. Antibody mixtures were then applied as follows:Primary A - Mix together KKX 6.1, 1 to 500 3% & horse serum and C-peptide, 1 to 500. Primary B - Mix together Human insulin 1 to 500 and glucagon 1 to 200, incubate for 2 hours and then Wash in PBS 10min×4. Secondary A - Add anti-mouse AF594 1 to 500 and anti-rat AF488 1 to 500. Secondary B - Add anti-guinea pig AF488 1 to 500 with anti-mouse AF594 1 to 500 incubate for 30min then wash 10min 4×. Slides were then stained with DAPI and coverslips mounted using prolong gold antifade (Life Technologies, Carlsbad, CA)
Parafin slides were deparaffinized through subsequent incubations in the following solvents (Xylene 5min 2× 100% ETOH 2min ×2 95% 2 min ×2 70% 2min ×2 d-water). Antigen retrieval was done by incubating sections for 30min in ice cooled PBS, and then blocking with 3% horse serum to block for 30 min. Antibody mixtures were then applied as follows:
5
Multimodal Immunofluorescence of Pancreatic Hormones
6
Tissue Characterization Reagents Protocol
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Collagenase NB 4G was purchased from SERVA Elektrophoresis GmbH (Heidelberg, Germany). Fluorescein isothiocyanate (FITC)-dextran 150,000, rhodamine 6G and Hoechst 33,342 were purchased from Sigma-Aldrich (Taufkirchen, Germany), ketamine (Ursotamin®) was purchased from Serumwerke Bernburg (Bernburg, Germany) and xylazine (Rompun®) was purchased from Bayer (Leverkusen, Germany). HepatoQuick® and DPO-α (Aranesp®) were purchased from Amgen (München, Germany). The antibody anti-CD31 (DIA310) was received from Dianova (Germany), the antibodies anti-insulin and the anti-myeloperoxidase (MPO) antibody (ab9535) from Abcam (Cambridge, UK), the antibody anti-GFP from Rockland Immunochemical Inc. (Limerick, USA) and anti‐Casp‐3 antibody from New England Biolabs (Frankfurt am Main, Germany).
7
Immunofluorescence analysis of pancreatic cells
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Pancreatic ductal cells were seeded on glass coverslips, fixed in 4% PFA for 15 min, and permeabilized with 1% triton X-100 for 10 min. They were then blocked in 4% BSA for 30 min at room temperature and incubated with primary antibodies (anti-CK19, Abcam, 1:200; PDX 1, Abcam, 1:100, anti-insulin, Abcam, 1:200; anti-glucagon, CST, 1:200) at 4°C overnight. Next, the cells were incubated with secondary antibodies conjugated with FITC or Cy5. The primary antibodies were replaced by PBS using a negative control. Positive cells were observed using a Leica TCS-SP8 SR confocal microscope.
8
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.
9
Quantifying Pancreatic β-Cell Proliferation
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Pancreatic tissue was immunostained using anti-Ki67 (BD Biosciences) and anti-insulin (Abcam) antibodies. Ki67+ β-cells were visualized by immunofluorescence microscopy and counted by a blinded observer (14 (link)). Insulin-positive cells colocalized with nuclear DAPI and Ki67 immunostaining were counted as proliferating β-cells. β-Cell area was determined by ImageJ software (National Institutes of Health) and calculated as insulin-positive area divided by total pancreas area.
10
Quantitative Pancreas Biopsy Analysis
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