Histology was performed on pancreatic frozen sections from healthy mice by staining for insulin and colocalizing it with Cy5.5 signal derived from the probe 1.5, 7.5, and 24 h after injection. Sections were stained with anti-insulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with FITC-labeled secondary goat anti-rabbit IgG (Vector Laboratories, Inc., Burlingame, CA) and counterstained with DAPI (Vectashield; Vector Laboratories). Pancreatic frozen sections from prediabetic and diabetic NOD mice were also stained using anti–GLP-1R antibody (Abcam) or anti-insulin antibody (Santa Cruz Biotechnology) and colocalized with Cy5.5 signal. Observers blinded to the treatments evaluated all tissue sections.
Anti insulin antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-insulin antibody is a laboratory reagent used to detect and measure insulin levels in biological samples. It binds specifically to insulin, allowing for its quantification using various immunoassay techniques.
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Lab products found in correlation
In situ cell death detection kit, by Roche (2 mentions)
Dfc310 fx, by Leica camera (2 mentions)
Alexa fluor 488 goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Apopercentage apoptosis assay kit, by Biocolor (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti α tubulin antibody, by Abcam (1 mentions)
Ecl kit, by Bio-Rad (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Merck Group (1 mentions)
Trans blot turbo mini nitrocellulose transfer pack, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Bz x700, by Keyence (1 mentions)
Fluoview version 2, by Olympus (1 mentions)
Fv1000 microscope, by Olympus (1 mentions)
Histofluid mounting medium, by Marienfeld (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Fluorescein isothiocyanate conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Ezrmi 13k, by Merck Group (1 mentions)
Anti phospho stat3 antibody, by Cell Signaling Technology (1 mentions)
17 protocols using anti insulin antibody
1
Pancreatic Insulin Localization
2
Immunohistochemical Quantification of Pancreatic and Hepatic Apoptosis
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Pancreases and livers were fixed in 4% paraformaldehyde solution for 24 h, embedded in paraffin, cut into 5 μm sections, and placed onto glass slides. The β-cell mass was identified by immunohistochemical localization of insulin with an anti-insulin antibody (Santa Cruz Biotechnology, Inc., CA, USA). For the analysis of cell apoptosis, pancreatic and liver sections were stained with the TUNEL (KeyGen Biotech. Co., Ltd., Nanjing, China). DNase I pretreated cells were used as the positive control.
3
Western Blot Insulin Detection
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Following electrophoresis on polyacrylamide gels, proteins were electro-transferred to nitrocellulose membrane (Trans-Blot Turbo Mini Nitrocellulose Transfer packs; Bio-Rad) using a Trans-Blot Turbo Transfer System (Bio-Rad). After protein transfer, the membranes were blocked for 1 h in 5% (w/v) milk in PBS at room temperature, and then immunoblotted in 5% milk and 0.1% Tween-20 in PBS with anti-insulin antibody produced in rabbit (SantaCruz, Dallas, TX, USA). Then, blots were incubated with HRP-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich), and immune reactive bands were detected using an enhanced chemiluminescence method (ECL kit; BioRad). Densitometry was analyzed by using the NIH Image (v1.63) software (National Institutes of Health, Bethesda, MD, USA). The pixel intensity for each region was calculated, the background was subtracted, and the protein expression results were normalized with respect to the bands of an anti-α-tubulin antibody (Abcam, Cambridge, UK) as loading control for each lane.
4
Pancreatic Beta Cell Mass Quantification
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The mice were sacrificed by cervical dislocation; the pancreas was immediately resected. The tissue was immediately fixed in 10% formalin at 4°C. For BCM analysis, 10 sets of serial formalin-fixed paraffin-embedded sections (4 mm per section; 100 mm between each set) were stained with rabbit polyclonal anti-insulin antibody (1:100; catalog no. sc-9168; Santa Cruz Biotechnology, Santa Cruz, CA, USA) as primary antibody; Alexa Fluor 488 goat anti-rabbit antibody (1:200; catalog no. A-11008; Thermo Fisher Scientific, Waltham, MA, USA) was used as a secondary antibody, as previously reported (18 (link), 20 (link)). Adjacent sections were also stained with hematoxylin and eosin. The pancreatic sections were analyzed using a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). BCM was calculated from histological sections using the following formula: (insulin-positive area/whole pancreas area) × pancreas weight (mg) (18 (link)–20 (link)).
5
Assessing Pancreatic Islet Viability
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Pancreatic islets were isolated from male C57BL/6 mice using the collagenase digestion method. Islet viability was evaluated as previously described.21 (link) Briefly, islets were incubated with cytokines and fixed overnight in a solution of 6.5% glutaraldehyde. Islet viability was determined by hematoxylin–eosin staining and labeling for anti-insulin antibody (Santa Cruz Biotechnology). Apoptosis was detected by the ApoPercentage Apoptosis Assay Kit (Biocolor, Newtownabbey, Ireland). This assay relies on the ApoPercentage dye that is selectively imported by apoptotic cells.22 Apoptotic cells developed red color following the intake of ApoPercentage dye and were detected under an inverted light microscope.
6
Histological Analysis of Mouse Tissues
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Mouse liver tissue, epididymal adipose and brown adipose were fixed in 10% neutral-buffered formalin, embedded in paraffin and sectioned at 5 μm. H&E staining was performed using standard techniques.
Pancreatic tissues were harvested, embedded in Tissue-Tek, and ‘snapfrozen' in dry ice and ethanol and stored at −80 °C. Cryostat sections (10 μm in thickness) were prepared, air-dried and fixed in ice-cold acetone for 15 min. Sections were blocked with 5% goat serum, stained with anti-Insulin antibody (Santa Cruz Biotechnology, Dallas, TX, USA), mounted with histofluid mounting medium (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and analysed with an Olympus FV1000 microscope (Olympus, Shinjuku-ku, Tokyo, Japan). Images were acquired with Olympus Fluoview Version 2.1 software.
Pancreatic tissues were harvested, embedded in Tissue-Tek, and ‘snapfrozen' in dry ice and ethanol and stored at −80 °C. Cryostat sections (10 μm in thickness) were prepared, air-dried and fixed in ice-cold acetone for 15 min. Sections were blocked with 5% goat serum, stained with anti-Insulin antibody (Santa Cruz Biotechnology, Dallas, TX, USA), mounted with histofluid mounting medium (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and analysed with an Olympus FV1000 microscope (Olympus, Shinjuku-ku, Tokyo, Japan). Images were acquired with Olympus Fluoview Version 2.1 software.
7
TUNEL Assay for Beta-Cell Apoptosis
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Islet-containing kidney tissues were embedded in optimum cutting temperature (OCT) compound (Tissue-Tek, CA, USA), and beta-cell apoptosis was examined in cryosections (5-µm thick) using an in situ cell death detection kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s protocol. Briefly, the tissue sections were permeabilized with 1% proteinase K for 15 min, rinsed with PBS, incubated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reagents for 1 h at 37 °C, and washed in PBS for 5 min. Then, tissues were stained with an anti-insulin antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), a fluorescein isothiocyanate-conjugated secondary antibody, and 4′-6-diamidino-2-phenylindole (Life Technologies, Carlsbad, CA, USA). Images were captured using a laser scanning confocal microscope (Carl Zeiss).
8
Histological Analysis of Transplanted Mouse Tissues
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Mice were killed under nonfasting conditions 65 days after transplantation. The graft-bearing kidney and pancreatic specimens were immediately fixed in 10% formalin solution, embedded in paraffin and cut into 5 μm sections. Specimens were stained with hematoxylin–eosin to identify morphological changes. For immunohistochemical study, tissue sections were treated using a microwave antigen retrieval procedure in 0.01 mol l−1 sodium citrate buffer. After blocking endogenous peroxidase, the sections were incubated with Protein Block Serum-Free (DAKO, Carpinteria, CA, USA) to block nonspecific staining, then with anti-insulin antibody (Santa Cruz Biotechnology), anti-betacellulin antibody (R&D Systems), or anti-von Willebrand factor (vWF) antibody (Bio-Rad, Raleigh, NC, USA). Peroxidase activity was detected with 3-amino-9-ethyl carbazole.
9
Liver and Pancreas Histology Assessment
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All the animals were killed by an intraperitoneal injection of pentobarbital sodium (30 mg/kg bodyweight) after 24 weeks, and tissue biopsies from liver and pancreas were performed. Liver samples were fixed overnight in 4% paraformaldehyde in phosphate buffer saline (PBS) at 4°C. Serial sections of the right lobes of the liver were stained with haematoxylin and eosin (H&E), periodic acid‐Schiff stain (PAS) and Masson's trichrome. Frozen sections were stained with Oil Red O to visualize lipid accumulation. Immunohistochemistry staining with an anti‐insulin antibody (Santa Cruz) was used to confirm the presence of β‐cells. Images were captured with a digital camera and analysed blindly by two observers.
10
Insulin Production Regulation in MIN6 Cells
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HuD isoform (2.5 μg) plasmids were transfected in MIN6 cells and after 24 h of recovery, the cells were treated with low and high glucose media for 30 min. The insulin production was estimated from culture supernatant by ELISA (Millipore EZRMI-13K) following the instruction manual and the intracellular insulin levels were also assessed by probing with anti-insulin antibody (Santa Cruz Biotechnology-9160).
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