Whole-mount immunohistochemistry was performed using mouse anti-rat nkx6.1 (Developmental Studies Hybridoma Bank) and rabbit anti-insulin (abcam) antisera. Goat antimouse IgG AlexaFluor 488 (Invitrogen), goat anti-rabbit IgG AlexaFluor 568 (Invitrogen) and goat anti-mouse IgG AlexaFluor 647 (Invitrogen) were used as secondary antisera. Briefly, embryos were fixed overnight in 4% paraformaldehyde (PFA) in phosphatebuffered saline (PBS). Fixed embryos were sequentially washed with PBS Triton 0.1%, then treated for 30 min to 2 h with PBS-Triton 1%, washed again with PBS-Triton 0.1% and incubated for 1h with PBS-T+BSA 5% before the incubation with the primary antisera, diluted 1:50 in PBS-T+BSA, overnight at 4°C. After extensive wash, embryos were incubated with the secondary antisera, overnight at 4°C, washed extensively and mounted in 50% glycerol in PBS. Embryos were stained with DAPI and 1,5 μm Z-series stacks were acquired on a Leica SP5 confocal microscope, using a 40x objective. Post-imaging analysis and quantifications were done in Fiji-ImageJ.
Rabbit anti insulin
Manufactured by Abcam
Sourced in United States
Rabbit anti-insulin is a primary antibody that specifically binds to the insulin protein. It can be used to detect and quantify insulin levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1 fluorescent microscope, by Zeiss (2 mentions)
Rat anti bromodeoxyuridine brdu, by Abcam (2 mentions)
Cy 2 coupled donkey anti rat igg antibody, by Jackson ImmunoResearch (2 mentions)
Cy 3 coupled donkey anti rabbit igg antibody, by Jackson ImmunoResearch (2 mentions)
Axio vision imaging software v 4, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Hoechst, by Merck Group (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Ultrasensitive mouse insulin enzyme linked immunosorbent assay elisa kit, by Mercodia (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Lowicryl resin, by Polysciences (1 mentions)
Mouse anti glucagon, by Abcam (1 mentions)
9 protocols using rabbit anti insulin
1
Whole-mount immunohistochemistry of embryos
2
Quantifying Pancreatic Beta Cell Proliferation
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Paraffin-embedded pancreatic sections were incubated with primary antibodies followed by visualization using fluorescent secondary antibodies as previously published [16 (link)]. Primary antibodies: rabbit anti-insulin at 1:500 (Abcam, Cambridge, MA) and rat anti-bromodeoxyuridine (BrdU) at 1:1000 (Abcam). Secondary Abs: Cy-2 coupled donkey anti-rat IgG antibody at 1:200 (Jackson Immuno Research, West Grove, PA); Cy-3 coupled donkey anti-rabbit IgG antibody at 1:200 (Jackson Immuno Research). Nuclei were visualized with 1 μg/ml 4’,6-diamidino-2-phenylindole (DAPI, Life Technologies, Carlsbad, CA). Images of pancreatic sections were recorded with Axio Observer Z.1 fluorescent microscope with Axio Vision Imaging Software v 4.7.1.0 (Carl Zeiss, Oberkochen, Germany). Total beta cell area was measured via summation of insulin positive area in a pancreatic section with maximum footprint for each mouse using Mosai X (scan mode: meander, autofocus per tile) in Axio Vision. BrdU positive nuclei were manually selected if 1) it was surrounded by insulin and 2) superimposed with DAPI to pick up a proliferating beta-cell. The number of BrdU positive nuclei was corrected for total beta cell area of each mouse.
3
Quantifying Pancreatic Beta Cell Proliferation
4
Characterization of Mouse GFP+ iPSC-derived β-cells
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The mouse GFP+-iPSC-derived β-like cells were characterized by immunofluorescence and glucose-stimulated insulin secretion. For immunofluorescence, cells were fixed by 4% paraformaldehyde (PFA) (Solarbio, Beijing, China) for 20 min, permeabilized by 0.1% Triton X-100 (Solarbio) for 10 min, and blocked with 5% bovine serum albumin (BSA) (Solarbio) for 30 min. After that, the cells were incubated with rabbit anti-insulin 1:100 (Abcam, Cambridge, MA, USA) or rabbit anti-C-peptide 1:100 (Abcam) overnight at 4 °C. The next day, the cells were washed 3 times with phosphate-buffered saline (PBS) and incubated with Alexa Flour 594-conjugated goat anti-rabbit secondary antibody 1:500 (Abcam) at room temperature for 1 h. Subsequently, the cells were stained with Hoechst (Sigma-Aldrich) for 15 min and washed with PBS 3 times. The cells were visualized by an Olympus fluorescence microscope (Olympus, Tokyo, Japan).
For a glucose-stimulated insulin secretion assay, the iPSC-derived β-like cells were exposed to a glucose gradient (0, 5, 15, 30, and 45 mM), and insulin secretion was measured by an ultrasensitive mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (Mercodia, Uppsala, Sweden).
For a glucose-stimulated insulin secretion assay, the iPSC-derived β-like cells were exposed to a glucose gradient (0, 5, 15, 30, and 45 mM), and insulin secretion was measured by an ultrasensitive mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (Mercodia, Uppsala, Sweden).
5
Histological Evaluation of Islet Grafts
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Histological examination was performed as previously described.19 Renal tissues containing islet grafts were cut and fixed with 10% formalin at room temperature for 24 h. Consecutive sections (5 um thick) of paraffin‐embedded renal tissues containing islet grafts were cut and subjected to immunohistochemical (IHC) staining. For IHC staining, sections were blocked with 1% BSA, and incubated with diluted primary antibodies including rabbit anti‐insulin (Abcam), then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody (DAKO) and finally stained with 3,3′‐diaminobenzidine (DAB) substrate and haematoxylin. Immunofluorescence (IF) was performed with goat anti‐mice insulin (Abcam) and rabbit anti‐mice CD31 (Abcam). The images of stained sections were acquired by fluorescence microscope (Carl Zeiss), and insulin and CD31 positive area were quantified using Image J software.
6
Ultrastructural Analysis of Insulin and Glucagon
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To analyze the granular ultrastructure, the cells were fixed with 1% glutaraldehyde and 1% PFA in 0.1 M sodium cacodylate buffer (pH 7.2) at 4 °C. Specimens were then fixed in 2% osmium tetroxide for 60 min at 4 °C. Dehydration of the fixed samples was performed, and the samples were transferred to Lowicryl resin (Polyscience, Niles, IL, USA). Samples were then sectioned (60 nm) with an ultramicrotome (UltracutUCT, Leica, Wetzlar, Germany) and collected on nickel grids. Post-embedding immunogold labeling was performed for insulin and glucagon labeling using the rabbit anti-insulin (Abcam), mouse anti-glucagon (Abcam), 5-nm colloidal gold conjugated to goat anti-rabbit IgG (Sigma-Aldrich), and 9–11-nm colloidal gold conjugated to goat anti-mouse IgG (Sigma-Aldrich). Following immunogold labeling, the sections were double-stained with 2% uranyl acetate for 20 min and lead citrate for 10 min. The sections were then viewed using TEM.
7
Characterization of Transplanted Liver Tissue
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After sacrificing the mice on days 7 and 14, the livers were removed and fixed with 4% formalin. A paraffin block was prepared and cut into 4-μm sections. The tissue slides were deparaffinized and dehydrated. Samples subjected to antigen retrieval were blocked with 3% bovine serum albumin. Immunohistochemistry was performed using primary antibodies for rabbit anti-PDX1 (dilution 1:200, Abcam) and rabbit anti-insulin (dilution 1:1000, Abcam). To confirm neovascularization in transplanted site, we also stained liver tissue using CD31 (dilution 1:1000, Abcam). A DAB Detection Kit (REAL EnVision detection system, DAKO, Glostrup, Denmark) was used for immunohistochemistry. To perform hematoxylin and eosin staining, samples were deparaffinized and dehydrated, followed by staining with hematoxylin (Sigma-Aldrich) and eosin (Sigma-Aldrich).
8
Immunohistochemical Analysis of Pancreatic Islet Cells
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FFPE pancreas serial cross-sections (4 μm thick) were deparaffinized and rehydrated. Heat-induced epitope retrieval was performed using Borg Decloaker (Biocare Medical, Pacheco, CA) according to the manufacturer's instructions prior to staining with 2 antibody panels: (i) rabbit anti-Ki67 (clone EPR3610, diluted 1:1000, RRID: AB_10562976; Abcam, Waltham, MA, USA), rabbit anti-insulin (clone EPR17359, diluted 1:2000, RRID: AB_2716761; Abcam), mouse anti-glucagon (clone K79bB10, diluted 1:1,000, RRID: AB_297642; Abcam) (Fig. 1A ); and (ii) rabbit anti-somatostatin (polyclonal, diluted 1:1,000, RRID: AB_2688022; Agilent Technologies, Inc., Santa Clara, CA, USA), rabbit anti-insulin (clone EPR17359, diluted 1:2,000, RRID: AB_2716761; Abcam), and mouse-pancreatic polypeptide (clone MM0858-31R25, diluted 1:750, RRID:AB_2904511; Abcam) (Fig. 1B ). Chromogen-based immunohistochemistry (IHC) staining was detected using a Mach2 Double Stain1/Mach2 Double Stain 2 HRP-AP Polymer Detection Kit according to the manufacturer's instructions (Biocare Medical, Pacheco, CA) with Betazoid DAB (Ki67 or somatostatin), Warp Red (insulin), and Ferangi Blue (glucagon or pancreatic polypeptide; all from Biocare Medical). Slides were counterstained with hematoxylin.
9
Histological Analysis of Pancreatic Tissue
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After the mice were sacrificed on day 60, the harvested tissues were fixed in 10% formalin solution for 24 h at 4 °C. A paraffin block was prepared with the fixed tissue and cut into 4 μM sections. The samples were deparaffinized, dehydrated, and subjected to staining with hematoxylin and eosin (Sigma Aldrich). Immunohistochemistry was performed using the primary antibodies of rabbit anti-PDX1 and rabbit anti-insulin (dilution 1:200, Abcam, Cambridge, UK). The sections (4 μM thickness) were deparaffinized, dehydrated through a graded alcohol series, blocked with hydrogen peroxide, and dried for 10 min at RT and 20 min in an incubator at 65 °C. An automated slide preparation system (Benchmark XT; Ventana Medical Systems Inc., Tucson, AZ, USA) with the OptiView DAB Detection Kit (Ventana Medical Systems, Tucson, AZ, USA) was used for the immunohistochemistry.
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