Whole pancreas was embedded in Tissue Tek (Sakura Fneteci), frozen, cut into sections (8 um). Histology slides containing sections from the pancreas were fixed with 4%paraformaldehyde, permeabilized with 0.01% Triton X-100.For histology, the slides from each group were stained with hematoxylin and eosin. For Immunofluorescent staining, nonspecific binding sites were blocked in 10% bovine serum in PBS at room temperature for 30 min. The slides were then incubated at 4 °C overnight with diluted primary antibodies and with Alexa Fluor labeled secondary antibodies at 37 °C for 60 mins in the dark followed by DAPI for 30 mins at room temperature in the dark. The slides were then rinsed with PBS and evaluated under a fluorescence microscope. The primary antibodies used were anti-insulin antibody (ab6995, Abcam), anti-glucagon antibody (ab8055, Abcam). The images were collected using a Nikon TE2000-U inverted fluorescence microscope (Nikon Co., Tokyo, Japan).
Ab8055
Manufactured by Abcam
Sourced in United Kingdom
Ab8055 is a recombinant monoclonal antibody that can be used as a tool for research applications. It is produced in a mammalian cell line and purified using protein A affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Ab6995, by Abcam (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Te2000 u inverted fluorescence microscope, by Nikon (1 mentions)
Ab103790, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ti e fluorescence microscope, by Nikon (1 mentions)
Horseradish peroxidase pod, by Roche (1 mentions)
Cm3050 cryostat, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Mouse anti insulin, by Abcam (1 mentions)
Dylight594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti c peptide, by Cell Signaling Technology (1 mentions)
Fluoroshield dapi, by Merck Group (1 mentions)
Dylight594 goat anti mouse, by Jackson ImmunoResearch (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
4 protocols using ab8055
1
Immunohistochemistry of Pancreatic Islet Cells
2
Pancreatic Cell Immunohistochemistry
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Whole 5% paraformaldehyde-fixed and paraffin-embedded samples of pancreas from Wistar and Goto-Kakizaki rats, typically of ages of 31, 52, and 79 weeks, were investigated using either monoclonal antibody mouse anti-insulin ab6995 (ABCAM, Cambridge, UK), diluted 1 : 100, or polyclonal antibodies such as rabbit anti-glucagon ab8055 (ABCAM), diluted 1 : 100, rabbit anti-somatostatin ab103790 (ABCAM), diluted 1 : 500, and rabbit anti-pancreatic polypeptide PA1-36141 (Pierce Biotechnology, Rockford, IL), diluted 1 : 1000. Secondary antibodies (Life Technologies), diluted 1 : 1000, were Alexa Fluor 568 Donkey anti-Mouse IgG, A10037 for red imaging, and Alexa Fluor 488 Donkey anti-rabbit IgG (H + L), A21206, for green imaging. Immunohistochemical samples were viewed by a motorized inverted fluorescence microscope Olympus IX-81 and Cell F software.
3
Multi-omic Colon and Ileum Analysis
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For detailed protocol, see Supplementary methods. Briefly, 12 μm sections of mouse colon and ileum were cut using a Leica CM3050 cryostat and mounted on Superfrost Plus slides (Thermo Fisher Scientific) and allowed to air-dry for an hour. Sections were hybridised with DIG-labelled Insl5 or Rxfp4 probe (1:100 each) overnight at 60°C, washed and incubated with horseradish peroxidase-(POD, 1:500, Roche) conjugated anti-DIG antibody before tyramide signal amplification with FITC-tyramide. Following in situ hybridisation, sections were blocked (2% BSA, 0.1% (v/v) Tween-20 in PBS), incubated with rabbit anti-proglucagon antibody (1:100, ab8055, Abcam) overnight, washed with TBST and incubated with anti-rabbit Alexa594 secondary antibody (1:500, Thermo Fisher Scientific). Fluorescent wide-field images were acquired by tile-scanning using a Nikon Ti-E fluorescence microscope with a 20× objective and photo-stitched during acquisition in NIS-Elements software (Nikon Instruments).
4
Immunostaining of 3D hAEC Spheroids
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3D hAEC spheroids were fixed with 2% Paraformaldehyde (PFA, Sigma-Aldrich) for 20 min at room temperature. After washing 2 × 10 min in PBS, spheroids were permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 10 min and then rinsed again 2 × 10 min in PBS. Antigen blocking was performed for 1 h at room temperature in blocking solution: 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA), 0.2% Triton X-100, 0.1% Bovine Serum Albumin (BSA) (Sigma-Aldrich). Primary and secondary antibodies were diluted in freshly prepared blocking solution. Primary antibodies were incubated at 8°C overnight and secondary antibodies for 40-50 min at room temperature. The following primary antibodies and dilutions were used: mouse anti-insulin 1:100 (Abcam ab46707); rabbit anti-glucagon (Abcam ab8055); rabbit anti-C-peptide 1:100 (Cell Signalling #4593); rabbit anti-CK19 1:100 (Thermo Scientific PA5-16726); mouse antilaminin 1:100 (Thermo Scientific MA1-21194). Secondary antibodies were: DyLight 594 goat antimouse 1:200 (Jackson Immuno Research Labs.); Alexa Fluor 488 goat anti-rabbit 1:400 (Molecular Probes); CF 488 goat anti-mouse 1:400 (Biotium); DyLight 594 goat anti-rabbit 1:200 (Thermo Scientific). Nuclei counterstaining was performed with Fluoroshield+DAPI (Sigma-Aldrich). Imaging
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