After fixation with 4% paraformaldehyde, sections were washed 3 times with PBS, and then permeabilized with 0.3% Triton X-100 at 37°C for 15 min. After rinsing 3 times with PBS, the sections were treated with goat serum for half an hour at 37°C. Then, they were incubated with an anti-MAP2 antibody (1: 200, Proteintech, 17490-1-AP) or an anti-SYN antibody (1: 200, Abcam, ab32127) overnight at 4°C. Sections were then incubated with fluorescent secondary antibody for 1 h at 37°C followed by counterstaining with DAPI for 15 min. Finally, labeling was visualized using a fluorescence microscope (Olympus FV1000) and analyzed using an Olympus Fluoview Ver.1.7a viewer.
Fluoview ver 1.7a viewer
Manufactured by Olympus
Sourced in Japan
The Fluoview Ver.1.7a viewer is a software application designed for the visualization and analysis of fluorescence microscopy images. It provides a platform for viewing and manipulating digital images captured using Olympus microscopy equipment.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 594, by Thermo Fisher Scientific (2 mentions)
Fv1000 fluoview camera, by Olympus (2 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
Glucagon, by Merck Group (2 mentions)
E cadherin, by Abcam (2 mentions)
H2dcfda, by BD (2 mentions)
Fv1000, by Olympus (1 mentions)
H2dcfda, by Olympus (1 mentions)
Facscalibur, by BD (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Confocal laser scanning biological microscope, by Olympus (1 mentions)
Hematoxylin and eosin, by Thermo Fisher Scientific (1 mentions)
Alexa 405, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Merck Group (1 mentions)
Nestin, by Abcam (1 mentions)
F4 80, by Merck Group (1 mentions)
Vimentin, by Abcam (1 mentions)
Hoechst 34580, by Thermo Fisher Scientific (1 mentions)
Tx 100, by Merck Group (1 mentions)
6 protocols using fluoview ver 1.7a viewer
1
Immunofluorescent Staining of MAP2 and SYN
2
Measuring Intracellular ROS Generation
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The fluorescent probe 2’,7’-dichlorofluorescin diacetate (H2DCFDA, Sigma-Aldrich, D6883) was used to measure the intracellular generation of ROS by N-PCNSs. Briefly, confluent HepG2 cells on coverslips (BD Biosciences) were incubated with 200 μg mL−1 N-PCNSs for 48 h. After washing with PBS, the cells were incubated with 10 μM H2DCFDA in serum-free DMEM for 20 min at 37 °C in the dark. The fluorescence intensities of H2DCFDA were measured by confocal laser scanning microscope (Olympus FluoView FV-1000, Tokyo, Japan). The quantitative analysis of the intensity of H2DCFDA was performed using Olympus FluoView Ver.1.7a Viewer. For flow cytometry analysis of ROS, after incubation with H2DCFDA, cells were measured immediately by flow cytometry (FACSCaliburTM, Becton Dickinson, Franklin Lakes, NJ, USA) with excitation at 488 nm and emission at 530 nm. Green mean fluorescence intensities were analyzed using FlowJo7.6 software (Tree Star, OR, USA).
3
Daphnetin Modulates NFAT and NF-κB Localization
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The nuclear distribution of NFAT and NF-κB were measured using immunofluorescense. Mouse CD3 T cells (2×106 cells/mL) in 2 mL of RPMI 1640 complete medium were incubated with 1 mL daphnetin (4, 8, 16 µg/mL) in 6-well plates at 37°C for 1 h followed by 0.5 h incubation with 1 mL of ConA (5 µg/mL), resulting in a final well volume of 4 mL per well. After over-night incubation in anti-NFAT2 antibody (1∶350) or anti-NF-κB antibody (1∶100), the cells were washed twice and incubated with Goat anti-mouse FITC-linked secondary for 1 h. Fluorescence images were visualized using an Olympus Confocal Laser Scanning Biological Microscope and analyzed using Olympus Fluoview Ver. 1.7a viewer software.
4
Comprehensive Pancreatic Histology Analysis
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For the histology of the pancreas, paraffin sections of 5 μm were stained with hematoxylin and eosin (Richard Allan Scientific, Kalamazoo, MI). For the immunofluorescence analysis, the frozen sections were incubated for 14 hours at 4 °C with antisera specific for insulin (1/150, guinea pig, Sigma), glucagon (1/2,000, mouse, Sigma), E-cadherin (1/100, rabbit, Abcam, San Francisco, USA), vimentin (1/100, rabbit, Abcam), vimentin (1/100, mouse, Sigma), MafB (1/200, rabbit, Bethyl Laboratories, Montgomery, USA), MafA (1/200, rabbit, Bethyl Laboratories), Pdx1 (1/50, goat, R&D system, Minneapolis, USA), Ngn3 (1/200, rabbit, Millipore), F480 (1/200, rabbit, Santa Cruz, Texas, USA), nestin (1/200, mouse, Abcam) and Ki67 (1/50, mouse, BD, San Diego, CA ). The slides were then incubated for 2 hours at room temperature with species-specific secondary antibodies (1:500; Alexa-594, Alexa-488 or Alexa-405; Invitrogen, Basel, Switzerland). The nuclei were visualized with DAPI (40, 6-diamidino-2-phenylindole) (Sigma). Images were captured with a Fluoview FV1000 camera (Olympus, Tokyo, Japan) and recorded on a computer using the Olympus Fluoview Ver.1.7a viewer.
5
Quantifying Apoptosis and TFEB Localization
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Cells were seeded on coverslips and were fixed with 4% paraformaldehyde for 30min at room temperature. After permeabilization with 0.2% TX-100 (Sigma, X100) for 10min at room temperature, cells were incubated with cleaved caspase 3 antibody (ab13847), cathepsin B antibody (ab33538) or TFEB antibody (sc-48784) for 2h at room temperature. After washing with PBS, cells were incubated with Rhodamine RedTM-X goat anti-rabbit IgG (Molecular Probes, R6394) and with Hoechst 34580 (Molecular Probes, H21486) for 1h. Immunofluorescence images were viewed under Olympus FluoView1000 (FV1000; Olympus) with identical acquisition parameters for the same image session and analyzed with Olympus FLUOVIEW Ver1.7a Viewer. Cleaved caspase 3 and TFEB nuclear staining were quantified with the ImageJ software. Briefly, nucleus of cell of interest was defined using the drawing tool. Mean fluorescence of the nuclear staining was obtained by selecting the “measure” option. After deducting any background staining, the mean fluorescence of at least 50 cells from each treatment was used for statistical analysis. Data is represented as mean +/- SEM (standard error of mean).
6
Immunofluorescence Analysis of Pancreatic Cells
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For the immunofluorescence analysis, the frozen sections were incubated for 14 h at 4°C with antisera specific for insulin (1/150, guinea pig; Sigma), glucagon (1/2,000, mouse; Sigma), glucagon (1/100, rabbit; Cell Signaling Technology, Danvers, MA, USA), Glut2 (1/100, mouse; Abcam, San Francisco, CA, USA), v‐maf musculopeoneurotic fiberosarcoma oncogene homologue A (MafA; 1/200, rabbit; Bethyl Laboratories, Montgomery, TX, USA), Pdx1 (1/50, goat; R&D System, Minneapolis, MN, USA), Ngn3 (1/200, rabbit; Millipore, St. Louis, MO, USA), E‐cadherin (1/100, rabbit; Abcam) and Ki67 (1/50, mouse; BD, Biosciences). The slides were then incubated for 2 h at room temperature with species‐specific secondary antibodies (1:500, Alexa‐594 or Alexa‐488; Invitrogen, Basel, Switzerland). β‐Cell apoptosis was determined using In Situ Cell Death Detection Kit (Roche, Basel, Switzerland). The nuclei were visualized with 40′,6‐diamidino‐2‐phenylindole (Sigma). Images were captured with a Fluoview FV1000 camera (Olympus, Tokyo, Japan) and recorded on a computer using the Olympus Fluoview Ver.1.7a viewer.
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