Whole pancreatic OPT, to 19 µm resolution, was performed as previously described (67 (link),70 (link)). Dual labelling for insulin and glucagon was performed using anti-insulin antibody (DAKO) and anti-glucagon antibody (Sigma) revealed using Alexa Fluor 568 and 680 (Invitrogen), respectively.
Anti glucagon antibody
Manufactured by Merck Group
Sourced in United Kingdom
The Anti-glucagon Antibody is a laboratory reagent used in research applications. It is designed to detect and quantify the presence of the glucagon hormone in biological samples. The antibody specifically binds to glucagon, allowing researchers to analyze its levels and distribution in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Anti insulin antibody, by Agilent Technologies (1 mentions)
Sp8 confocal system, by Leica (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa coupled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti swine insulin antibody, by Agilent Technologies (1 mentions)
4 protocols using anti glucagon antibody
1
Whole Pancreatic Optical Projection Tomography
2
Quantifying Pancreatic Alpha Cell Mass
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The pancreas was embedded in paraffin and sectioned to a thickness of 4 μm. IHC staining was performed with the anti-glucagon antibody (Sigma-Aldrich), and the α cell mass was then estimated by point counting.12 (link) In brief, the point numbers of glucagon-stained areas were counted at a magnification of 400× using a 48-point grid. The relative α cell area was calculated by dividing the point number of the glucagon-stained area by the point number over the entire pancreatic tissue sample on the same section. Two sections, separate from each other by 20 μm per mouse, were used for the mean relative area determination. The average number of islets was 20 ± 10 per section. The α cell mass was then estimated by multiplying α cell relative area by the pancreas weight.
3
Digoxigenin-Labeled RNA Probes for Whole-Mount In Situ Hybridization and Immunofluorescence
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The digoxigenin-labeled antisense RNA probes were synthesized in vitro by T3 RNA polymerase (Beyotime D7066). The whole-mount in situ hybridization was performed as described previously (23 (link)).
The whole-mount immunofluorescence was carried out as described by Jennifer et al. (52 (link)). The immunofluorescence was performed following in situ hybridization. In brief, the larvae were incubated with antiglucagon antibody (Sigma G2654) at 4 °C overnight, then washed with PBST (0.1% tween-20 in PBS). Alexa Fluor 488 secondary antibody (Thermo Fisher A11001, 1：1000) was used for detection following incubation for 2 h at room temperature (RT). The images were captured by Leica SP8 confocal system.
The whole-mount immunofluorescence was carried out as described by Jennifer et al. (52 (link)). The immunofluorescence was performed following in situ hybridization. In brief, the larvae were incubated with antiglucagon antibody (Sigma G2654) at 4 °C overnight, then washed with PBST (0.1% tween-20 in PBS). Alexa Fluor 488 secondary antibody (Thermo Fisher A11001, 1：1000) was used for detection following incubation for 2 h at room temperature (RT). The images were captured by Leica SP8 confocal system.
4
Immuno-fluorescent Labeling of Pancreatic Islet Cells
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Cells were fixed in 3.7% paraformaldehyde and permeabilised in 0.1% Triton X-100 before immunostaining with a polyclonal anti-swine insulin antibody (1:200, DakoCytomation, Ely, UK), anti-glucagon antibody (G2654, Sigma) and anti-rodent human/rodent ZnT8 antibody overnight at 4°C. Alexa-coupled secondary antibodies (Invitrogen) were used to reveal the primary antibody staining. Confocal imaging was performed as described elsewhere [15 (link)].
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