Immunohistochemistry of frozen or paraffin-embedded sections of pancreas, human islets, and human islet-like organoids (HILOs) was performed with antibodies to insulin (1/100, Abcam ab7842), c-peptide (1/100, Abcam ab30477), glucagon (1/100, Abcam ab10988), somatostatin (1/100, Abcam ab103790), pancreatic polypeptyde (1/100, Abcam, ab113694), NKX2–2 (1/100, DSHB, 74.5A5), NKX6–1 (1/100, DSHB, F55A12), MAFA (1/100, Abcam, ab26405), MAFB (1/100, Abcam, ab66506), PDX-1 (1/100, R&D, AF2419), CHGA (1/100, Abcam, ab15160), SYNAPTOPHYSIN (1/100, Biogenex, MU363-UC) and PD-L1 (1/100, Abcam, ab20592). For MAFA and MAFB, signal amplification was performed using TSA-Cy3 kit (Akoya Biosciences, SAT704A001EA). Secondary antibodies were coupled to Alexa 568, 647 (Life Technologies) and visualized by confocal microscopy (ZEISS) or fluorescence microscopy. Hoechst 33342 (Thermo Scientific, 62249, 1μg/ml final concentration) was used for nuclear staining.
Ab26405
Manufactured by Abcam
Sourced in United Kingdom
Ab26405 is a lab equipment product. It is a device designed for laboratory use. The core function of this product is to perform specific tasks within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab7842, by Abcam (2 mentions)
Ab30477, by Abcam (2 mentions)
Ab103790, by Abcam (2 mentions)
Ab66506, by R&D Systems (2 mentions)
Ab15160, by BioGenex (2 mentions)
Ab20592, by Abcam (2 mentions)
Ab10988, by Abcam (2 mentions)
Af2419, by Abcam (2 mentions)
Mu363 uc, by Abcam (2 mentions)
Tsa cy3 kit, by Akoya Biosciences (2 mentions)
Alexa 647, by Thermo Fisher Scientific (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Ab30477, by BD (2 mentions)
Ab82270, by Abcam (2 mentions)
Ab66506, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
G2654, by Merck Group (1 mentions)
Cell recovery solution, by Corning (1 mentions)
Ab30788, by Abcam (1 mentions)
7 protocols using ab26405
1
Multiparametric Islet Immunophenotyping
2
Immunofluorescence Analysis of Expanded Islet Clusters
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Expanded islet clusters were harvested using cell recovery solution (Corning, 354253) and fixed in 4% paraformaldehyde for 30 min at room temperature, followed with blocking and permeabilizing in PBS with 0.5% Triton X-100 (Solarbio, T8200) and 5% donkey serum (Solarbio, SL050) for 30 min at room temperature. Then, samples were incubated with primary antibody at 4 °C overnight, followed by incubation with secondary antibody for 2 h at room temperature. DAPI (Beyotime, C1002) was used to stain the nucleus and find islets. The following antibodies were used for immunofluorescence: anti-insulin (1:200, sc-9168; Santa), anti-somatostatin (1:600, ab30788; Abcam), anti-glucagon (1:200, G2654; Sigma). anti-PDX1 (1:200, ab47267; abcam), anti-SOX9 (1:200, ab185966; abcam), anti-NKX6.1 (1:200, ab221549; abcam), anti-MAFA (1:200, ab26405; abcam), anti-KI67 (1:200, D3B5; Cell Signaling Technology). Imaging of the expanded islet clusters was performed on Zeiss LSM 780 and processed using ImageJ or Adobe illustrator software.
3
Immunohistochemical Characterization of Pancreatic Tissue
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Serial sections from archival pancreatic tissue were subjected to MAFA immunohistochemistry as well as insulin and glucagon staining. The result was evaluated and compared to that of normal human pancreas by an experienced pathologist. To achieve comparability, MAFA immunohistochemistry was performed using the same MAFA antibody (Anti-MafA antibody ab26405, Abcam, Cambridge, UK, RRID: AB_776146; dilution: 1:2000) as Iacovazzo et al. [12 (link)] following the manufacturer’s instructions. After deparaffinization, hydration, and heat-induced epitope retrieval, incubation steps were carried out in an Autostainer 480-A (Thermo Fisher Scientific, Dublin, Ireland) using the Dako EnVision™ FLEX HRP/Dab (Agilent, Santa Clara, CA, USA) detection system with signal amplification by EnVision™ FLEX + Rabbit (LINKER) (Agilent, Santa Clara, CA, USA).
Standard staining/immunohistochemisty protocols of the Institute of Pathology (University Medical Center Mainz) were used to detect the neuroendocrine/proliferation markers chromogranin, synaptophysin, insulin, glucagon, and KI-67 in formalin-fixed, paraffin-embedded pancreatic tissue sections from patients IV3 and IV4 following resection.
Standard staining/immunohistochemisty protocols of the Institute of Pathology (University Medical Center Mainz) were used to detect the neuroendocrine/proliferation markers chromogranin, synaptophysin, insulin, glucagon, and KI-67 in formalin-fixed, paraffin-embedded pancreatic tissue sections from patients IV3 and IV4 following resection.
4
Multiparametric Islet Immunophenotyping
5
Multicolor Flow Cytometry Analysis of Pancreatic Endocrine Cells
6
Multimarker Immunostaining of Pancreatic Cells
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INS-1 832/13 cells were washed twice in PBS and fixed with 3% PFA in PBS for 30 min at 37 °C followed by washing 3 × 10 min with PBS before permeabilization for 30 min with BD Phosphflow Perm buffer III (BD Biosciences). Samples were blocked using 5% normal donkey serum in PBS for 30 min at room temperature. Cells were incubated overnight at 4 °C with antibodies against Cav1.2 (1:200, code C1603, Sigma-Aldrich), Cav1.3 (1:100, code ACC 005, Alomone Labs), MafA (1:100, code ab26405, Abcam), MafB (1:100, code IHC00351, Bethyl Labs), Pax6 (1:100, code MAB2237, Millipore) or Nkx6.1 (1:100, code AF5857, R&D Systems) diluted in the blocking solution. All antibodies were from rabbit except Nkx6.1, which was generated in mouse. Immunoreactivity against the rabbit antibodies was detected by a Delight 488-conjugated antibody (1:400, code 715-545-150, Jackson ImmunoResearch). A Cy5-conjugated antibody (1:400, code 715-175-150, Jackson ImmunoResearch) was used to detect the Nk × 6.1 antibody. Cell nuclei were counterstained with Hoechst 34580 (1:500, Life Technologies). Immunofluorescence images were acquired by confocal microscopy and Zen software (Carl Zeiss).
7
Multicolor Flow Cytometry Analysis of Pancreatic Endocrine Cells
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Clusters at indicated stages were dissociated with TrypLE (GIBCO) with 20μg/ml DNase for 12 min at 37°C and then fixed with 4% PFA for 10 min at room temperature. Clusters were then permeabilized with 0.2 % Triton X for 10 min, blocking with 10% goat serum for 30 min and stained for various intracellular markers with antibodies, c-peptide, (1/100, abcam, ab30477), PDX-1 (1/100, BD, 562161), NKX6–1 (1/100, BD, 563338), Chromogranin A (1/100, BD, 564583), MAFA (1/100, abcam, ab26405), MAFB (1/100, abcam, ab66506), Glucagon (1/100, abcam, ab82270), Somatostatin (1/100, abcam, 108456) for analysis BD Biosciences LSRII. Data were analysed by FlowJo software. Secondary antibodies for c-peptide, Glucagon and Somatostatin were coupled to Alexa 647 (Life Technologies).
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