Mouse pancreases were dissected and fixed in 4% formaldehyde at 4 °C for 12 h before embedding in paraffin62 (link). Two-four-micrometer sections were deparaffinized, rehydrated, and incubated overnight at 4 °C with anti-PDX-1 (Abcam; #47267), anti-Glut2 (Chemicon; #07-1402), anti-Ki67 (Dako; #M7249), anti-phospho-Histone H3 (Ser10; Merck #06-570), and anti-NKX6.1 (DSHB, University of Iowa #F55A1266 (link)) in combination with TSA (Invitrogen #T30955), or for 2 h at room temperature with anti-insulin (Dako; A0546) antibodies (all at a dilution of 1:100, except anti-PDX-1, which was diluted 1:400) followed by fluorescein isothiocyanate (FITC)- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Slides were mounted with Vectashield with 4′6-diamidino-2-phenylindole (DAPI) (Vector Labs). β-cell apoptosis was analyzed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique according to the manufacturer’s instructions (In Situ Cell Death Detection Kit, TMR red; Roche) and double stained for insulin. Fluorescence was analyzed by using a Nikon MEA53200 (Nikon GmbH, Dusseldorf, Germany) microscope, and images were acquired by using NIS-Elements software (Nikon).
Anti pdx1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-Pdx1 is a primary antibody that recognizes the Pdx1 protein. Pdx1 is a homeodomain-containing transcription factor that plays a crucial role in pancreatic development and function. This antibody can be used in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and study the Pdx1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti insulin, by Agilent Technologies (3 mentions)
Anti glut2, by Merck Group (3 mentions)
Fluorescein isothiocyanate fitc or cy3 conjugated secondary antibodies, by Jackson ImmunoResearch (3 mentions)
Nis elements software, by Nikon (3 mentions)
Vectashield with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (3 mentions)
Mea53200, by Nikon (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti p mst1, by Cell Signaling Technology (2 mentions)
Anti mouse anti ki67, by BD (2 mentions)
Anti glucagon, by Agilent Technologies (2 mentions)
Anti bim, by Cell Signaling Technology (2 mentions)
Anti ki67, by Agilent Technologies (1 mentions)
Anti phospho histone h3 ser10, by Merck Group (1 mentions)
Anti foxo1, by Santa Cruz Biotechnology (1 mentions)
Anti oct4, by Abcam (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti insulin, by Abcam (1 mentions)
Anti ngn3, by Abcam (1 mentions)
Anti arginase 1, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Abcam (1 mentions)
13 protocols using anti pdx1
1
Pancreatic Immunohistochemistry of Mouse
2
Islet Inflammatory and Dedifferentiation Analysis
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Islets were harvested, fixed in 4% paraformaldehyde, and immersed in 30% sucrose solution to be embedded in a frozen block. Inflammatory markers and dedifferentiation markers were detected in cell sections by immunofluorescence. Anti-FoxO1 (Santa Cruz, Dallas, Texas, USA), anti-cleaved caspase 3 (Cell Signaling, Danvers, MA, USA), anti-Oct4 (Abcam, Cambridge, UK), anti-NGN3 (Abcam), anti-A11 oligomer (Invitrogen), anti-Pdx1 (Abcam), anti-IL-1β (Abcam), and anti-insulin (Abcam) were used as primary antibodies. M1/M2 macrophage markers were identified using the antibodies of M2 macrophage differentiation markers (anti-arginase-1; Cell Signaling) and M1 macrophage differentiation markers (CD80; Abcam). Sections were incubated with primary antibody diluted in blocking buffer overnight at 4°C. Secondary antibodies (Alexa Fluor® 488-conjugated goat anti-rabbit, Alexa Fluor® 488-conjugated goat anti-mouse, and Alexa Fluor® 594-conjugated goat anti-rabbit; Abcam) were added and incubated for 1 h at room temperature. Counterstaining was performed using DAPI (1 : 10,000). Images were obtained from each section using a fluorescent microscope (Olympus, Shinjuku, Tokyo, Japan).
3
Comprehensive Pancreatic Tissue Analysis
4
Immunofluorescence Characterization of Isolated Cells
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The expression of cytokeratin-8 (CK-8), epithelial cell adhesion molecule (EpCAM) and pancreas/duodenum homeobox protein-1 (PDX-1) in the isolated primary cells was evaluated by immunofluorocytochemistry. For immunofluorescence labeling, one drop of cell suspension at a low density was dripped on the cover glass pretreated with poly-L-lysine, and then subjected to incubation at 37°C for 2–4 h, which can make the cells fully adherent. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then permeabilized with 2 mg/l to 0.03 mg/l of Triton X-100 in PBS. The nonspecific binding sites were blocked by incubating with 10 mg/l bovine serum albumin (BSA) in PBS for 45 min at room temperature. Slides were then incubated overnight at 4°C with the following primary Abs: anti-CK-8 (1:500, Abcam), anti-PDX1 (1:500, Abcam), anti-EpCAM (1:500, Santa Cruz Biotechnology), and the negative control was incubated with 1X PBS. After incubation, the slides were washed with PBS and further incubated with the donkey anti-mouse, donkey anti-goat, donkey anti-rabbit secondary Ab (dilution ratio 1:200, Life Technologies) for 2 h at room temperature. After extensive washes, the slides were mounted in glycerine (Beyotime) and observed using a confocal laser scanning microscope (Radiance Plus; Bio-Rad Laboratories, Milan, Italy).
5
Immunofluorescence Staining of Cultured Cells
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Cultured cells were fixed with 4% paraformaldehyde at room temperature for 15 min and blocked with blocking solution (DPBS with 10%(v/v) donkey serum and 0.3% Triton-100). Then the cells were incubated with primary antibodies overnight at 4 °C, followed with washing and staining with secondary antibodies for 2 h in dark at room temperature, and then counterstained with 5 μg/ml 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma, Cat#10236276001) for 10 min. Cells were visualized and imaged using fluorescence microscopy (Olympus). The antibodies for immunofluorescence assay used in present work include: anti-PAX6 (1:200, Biolegend, Cat#l901301), SOX1 (1:200, Cell Signaling Technology, Cat#4195S), anti-OCT4 (1:200, Santa Cruz biotechnology, Cat#sc-5279), anti-NANOG (1:200, Cell Signaling Technology, Cat#4903), anti-PDX1 1:200, abcam, Cat#ab47267), anti-T/Brachyury (1:200, Cell Signaling Technology, Cat#81694S), anti-TUJ1 (1:200, SIGMA, Cat#T3952).
6
Protein Expression Analysis in Pancreatic Tissues
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The pancreatic tissue samples (~30 mg) at the age of 12-week (n = 3 /group) were prepared by homogenization in a Radio Immuno Precipitation Assay (RIPA) buffer with protease inhibitors and centrifuged at 12,000 rpm, 4o C for 15 min. The protein concentrations were quantified by modified BCA protein assay (Beyotime, China). 100 μg cell lysates from each sample were separated by 12.5% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dried milk powder in TBST buffer for 2.5 h and were incubated with anti-Pdx1 (Abcam, USA) (1:500), anti-NeuroD1 (CST, Germany) (1:1000) and anti-pNeuroD1 (CST, Germany) (1:1000) overnight at 4o C. Anti-β-actin (Sigma, USA) (1:1000) was used as an internal control. The membranes were developed using ECL reagents (Millipore, Germany) and proteins were finally detected by a chemiluminescence detection system.
7
Immunohistochemical Analysis of Pancreatic Insulin and PDX-1
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Mouse pancreas was fixed in 10% neutral‐buffered formalin for 72 h, the tissue was then embedded in paraffin, and cut into 5‐μm sections. Immunohistochemistry was performed on a Leica Bond‐Max System (Leica Biosystems, Melbourne, Australia) using the Bond Polymer Refine Detection Kit (Leica Biosystems, Melbourne, Australia). Anti‐insulin (1:500, Invitrogen, Carlsbad, CA) antibodies were used followed by 30 min with HRP‐conjugated secondary antibody (1:500, MilliporeSigma, Burlington, MA). Anti‐PDX‐1 (1:500, Abcam, Cambridge, MA) antibodies were used for overnight incubation at 4°C. 3,3′‐Diaminobenzidine (DAB) was used for detection. Hamamatsu NanoZoomer digital slide scanner (Hamamatsu, Bridgewater, NJ) was used for imaging. For the determination of insulin‐positive area, four mice per group were used, and data were analyzed as the ratio of insulin‐positive area to total pancreatic tissue area. For PDX‐1, data were expressed as the ratio of nucleus PDX‐1‐positive area to total islet area. The sections were analyzed and quantified using Fiji Image J software (Schindelin et al, 2012 ).
8
Immunofluorescence Analysis of Pancreatic Tissues
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Pancreatic tissues were processed as previously described 66 (link). In brief, mouse pancreases were dissected and fixed in 4% formaldehyde at 4°C for 12h before embedding in paraffin. Human and mouse 4-μm sections were deparaffinized, rehydrated and incubated overnight at 4°C with anti-insulin (Dako), anti-P-MST1 (Cell Signaling), anti-Bim (Cell Signaling), anti-PDX-1 (abcam), anti-glucagon (Dako), anti-glut2 (Chemicon) and anti-mouse anti-Ki67 (BD Pharmingen) antibodies followed by fluorescein isothiocyanate (FITC)- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Slides were mounted with Vectashield with 4′6-diamidino-2-phenylindole (DAPI) (Vector Labs). beta-cell apoptosis for mouse sections or primary islets cultured on ECM dishes was analyzed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique according to the manufacturer’s instructions (In Situ Cell Death Detection Kit, TMR red; Roche) and double stained for insulin. Fluorescence was analyzed using a Nikon MEA53200 (Nikon GmbH, Dusseldorf, Germany) microscope and images were acquired using NIS-Elements software (Nikon).
9
Immunofluorescence Analysis of Pancreatic Tissues
10
Flow Cytometry for Cell Characterization
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Differentiated cells were incubated with Accutase (eBioscience) at 37 °C for 10 min to dissociate them. After the cells were centrifuged at 300 × g for 5 min, the pellet was resuspended in FACS buffer (PBS containing 1% FBS). The dissociated cells were incubated with anti-CXCR4 (BD Biosciences, Bedford, MA, USA) and anti-PDX1 (Abcam) at 4 °C for 30 min, followed by an incubation with Alexa Fluor 488-conjugated donkey secondary antibodies (Invitrogen) at 4 °C for 15 min. Then, the cells were washed 5 times with FACS buffer. The samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences). Data were evaluated using FlowJo software (Tree Star, Ashland, OR, USA).
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