The pancreas was extracted from mice and fixed overnight with 4% formalin, embedded in paraffin and microsectioned. Next, pancreas sections were stained using a polyclonal guinea-pig anti-insulin antibody (Dako-Agilent Technologies, CA, USA), followed by secondary incubation with an Alexa Fluor 555 anti-guinea-pig antibody (Invitrogen, CA, USA). For amyloid staining, pancreas sections were then incubated in 0.5% Thioflavin S (Sigma-Aldrich) solution for 2 min and rinsed twice with 70% ethanol. Nuclei were stained using Hoechst 33342. Fluorescence images were obtained using Leica LAS Image Analysis software. ImageJ version 1.49 software (National Institutes of Health) was used to determine the islet area and Thioflavin S staining area. Percentage of amyloid severity related to total islet area and amyloid prevalence related to total number of islets were calculated.
Polyclonal guinea pig anti insulin antibody
Manufactured by Agilent Technologies
Sourced in United States, Germany
Polyclonal guinea pig anti-insulin antibody is a laboratory reagent used to detect and measure insulin levels in biological samples. It is produced by immunizing guinea pigs with insulin, resulting in a mixture of antibodies that recognize different epitopes on the insulin molecule.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Methylbutane, by Merck Group (2 mentions)
Tcs sp5 confocal microscopy, by Leica (2 mentions)
Rat anti mouse cd16 cd32, by BD (2 mentions)
Alexafluor 647 conjuaged goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Alexafluor 555 conjugated anti guinea pig antibody, by Thermo Fisher Scientific (2 mentions)
Las image analysis software, by Leica (1 mentions)
Thioflavin s, by Merck Group (1 mentions)
Dm2500 microscope, by Leica (1 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 affinipure donkey anti guinea pig igg h l, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Power block, by BioGenex (1 mentions)
Alexa fluor 594 anti mouse, by Thermo Fisher Scientific (1 mentions)
Monoclonal mouse anti glucagon antibody, by Merck Group (1 mentions)
Alexafluor488 anti guinea pig, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Irdye 680cw goat anti mouse igg, by LI COR (1 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (1 mentions)
Donkey anti guinea pig secondary antibody, by Jackson ImmunoResearch (1 mentions)
9 protocols using polyclonal guinea pig anti insulin antibody
1
Quantifying Pancreatic Islet Amyloidosis
2
EdU Labeling of Proliferating NPICCs
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A Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific, Germering, Germany) was used to perform 5-Ethynyl-2′deoxyuridine (Edu) labeling. Briefly, NPICCs were cultured in B-IC medium in 6-well plates for six days in the presence of butyrate (1000 µM). EdU (10 µM) was added to the medium during the last 72 h. Then islets were embedded in Histogel and paraffin, sectioned and used for EdU staining according to the manufacturer’s instructions. Tissues were permeabilized with 0.5% Triton X-100 for 20 min and incubated with the Click-iT reaction cocktail for 30 min at room temperature. Slices were stained with polyclonal guinea pig anti-insulin antibody (diluted 1:300, Agilent-Dako, Frankfurt, Germany) for 1 h, and secondary antibody Alexa Fluor® 594 AffiniPure donkey anti-guinea pig IgG (H+L) for 45 min (1:1000, Jackson ImmunoResearch, Cambridgeshire, UK). Images were taken and quantified by Leica DM2500 microscope (Leica, Wetzlar, Germany) and Image J software (ImageJ 1.52a, http://rsb.info.nih.gov/ij/download.html , accessed on 23 April 2018). At least 1000 cells per each NPICC preparations were analyzed to count Edu labelled cells (n = 6).
3
Immunofluorescent Staining for Islet Hormones
4
Quantifying Islet Cell Populations
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Paraffin-embedded paraformaldehyde-fixed pancreases were sectioned in 200-μm increments and stained with polyclonal guinea pig anti-insulin antibody (1:1000, Dako, Jena, Germany) and mouse monoclonal anti-glucagon antibody (1:2000, Sigma–Aldrich). Secondary antibodies used were Alexa Fluor™ 488 anti-guinea pig and Alexa Fluor™ 594 anti-mouse (Thermo Fisher Scientific, Waltham, MA, USA), essentially as described [24 (link)]. Islet size was determined by measuring the insulin and glucagon area of 50 islets per mouse.
5
Effects of NI-203.26C11 on Pancreatic Insulin
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Example 4
To test the effect of the NI-203.26C11 on β-cells in hIAPP transgenic mice, pancreatic insulin, islet area, and insulin secretion was measured after a once-weekly treatment with recombinant mouse chimeric NI-203.26C11 antibody in hIAPP transgenic mice (tg NI-203.26C11-ch, n=23; 10 mg/kg i.p. for 12 weeks); see
In brief, paraffin-embedded sections were labeled with polyclonal guinea pig anti-insulin antibody (1:5; Dako, USA) and subsequently by TRITC-labeled donkey anti-guinea pig secondary antibody (1:500; Jackson ImmunoResearch Laboratories, USA). Stained samples were cover-slipped with Tris-buffered glycerol (a 3:7 mixture of 0.1 M Tris-HCl at pH 9.5 and glycerol supplemented with 50 mg/mL n-propyl-gallate). Quantification of the insulin-positive area in relation to the pancreas and islet area, mean islet area and islet density is described in the methods section.
The results showed pancreatic insulin (insulin-positive area in % pancreas area and % islet area; top left and top middle panels), islet area (mean islet area; top right panel) and insulin secretion (plasma insulin levels; bottom left panel) was increased compared with hIAPP transgenic mice receiving PBS (tg PBS, n=28). However, the islet density and pancreatic mass were unchanged after NI-203.26C11-ch treatment (bottom middle and bottom right panel, respectively;
6
Immunofluorescence and Western Blot Antibodies
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The following primary antibodies were used for immunofluorescence and western blot analysis: polyclonal guinea pig anti-insulin antibody (1:200, Dako, Carpentaria, CA, United States); mouse monoclonal anti-syntaxin-1A (1:1000, SySy); mouse monoclonal anti-SNAP-25 (1:1000, SySy); rabbit monoclonal anti-VAMP2 (1:1000, Abcam); rabbit monoclonal anti-tubulin (1:1000, Abcam). The secondary antibody was DyLight 488 goat anti-guinea pig IgG (1:500, Thermo); IRDye 800CW goat anti-rabbit IgG (1:1000, LI-COR Biosciences); and IRDye 680CW goat anti-mouse IgG (1:1000, LI-COR Biosciences).
7
Pancreatic Islet Cell Analysis
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Immunostaining was performed on formalin-fixed pancreatic tissue sections for insulin using guinea pig anti-insulin polyclonal antibody (Dako) and donkey anti-guinea pig secondary antibody conjugated with FITC (Jackson ImmunoResearch). Parallel sections were quantified based on DAPI staining for nuclei (blue) and insulin (green). Fluorescent images were acquired on a ZEISS inverted LSM700 microscope, using ZEN-lite digital imaging software (Carl Zeiss, Oberkochen, Germany) for processing. The fraction of beta cells represents the percentage of insulin positive cells in relation to the total number of cells per islet. The islets were from pancreata of mice with acute (n = 11) and progressive (n = 12) onset of diabetes and counted from 4 slides from each mouse at different section levels.
8
Islet Cryopreservation and Immunofluorescence Staining
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After the observation period of 200 days, long-term islet bearing kidneys were snap frozen in OCT compound (Sakura Tissue-Tek) by submerging in methyl butane (Sigma) on dry ice. Tissues were cut in 10 µm-thick slices using a Bright OTF5000 cryomicrotome (Rose Scientific) and put on frosted slides for staining. Slides were fixed in 4% paraformaldehyde, incubated in 0.5% Triton X-100, and blocked in 0.1% bovine serum albumin, 5% goat serum, and rat anti-mouse CD16/CD32 (BD Pharmingen). Staining was performed using rabbit anti-glucagon monoclonal antibody (Cell Signaling 8233S, 1:100) and guinea pig anti-insulin polyclonal antibody (Dako A0564, 1:100) as primary antibodies, followed by washing and staining with AlexaFluor-647-conjuaged goat anti-rabbit antibody (Life Technologies A21244, 1:100) and AlexaFluor-555-conjugated anti-guinea pig antibody (Invitrogen A21435, 1:300). Hoechst 33342 (Molecular Probes H3570, 1:25) was used to stain DNA. Fluorescent images were obtained using a Leica TCS SP5 confocal microscopy under 10X magnification.
9
Islet Cryopreservation and Immunofluorescence Staining
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