HBcAg expression in the HBV-infected cells was determined by a previously described IFA with modification [60 (link)]. Briefly, HepG2NTCP cells were seeded at 2 × 105 cells/well in 24-well cell culture plates with coverslips coated with collagen. Cells were infected with HBV as described above. Cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, followed by cell permeabilization with 0.1% Triton X-100 for 5 minutes and blocking with 3% BSA at 4°C overnight. HBcAg was stained with a HBc-specific monoclonal antibody C1-5 (100-fold dilution) at 4°C overnight and the secondary goat anti-mouse IgG conjugated with Alera Fluro 594 (2000-fold dilution) at room temperature for 2 h. Coverslips were mounted to glass slides using Prolong Diamond Antifade Mountant with DAPI (Invitrogen). Fluorescence images were taken with Keyence BZ-X800E fluorescent microscope.
Bz x800e fluorescent microscope
Manufactured by Keyence
The BZ-X800E is a fluorescent microscope designed for high-performance imaging. It features a motorized focus, LED illumination, and a high-resolution camera for capturing detailed images of fluorescently labeled samples. The core function of the BZ-X800E is to provide researchers with a tool for visualizing and analyzing fluorescent biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Px330 lmna grna1, by Addgene (2 mentions)
Nucred live 647 readyprobes reagent, by Thermo Fisher Scientific (2 mentions)
Jetoptimus, by Polyplus Transfection (2 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Ab125212, by Abcam (1 mentions)
Ab178847, by Abcam (1 mentions)
Ab104225, by Abcam (1 mentions)
Ab6326, by Abcam (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Evos fluorescent microscope, by Thermo Fisher Scientific (1 mentions)
4 protocols using bz x800e fluorescent microscope
1
Quantifying HBV Core Antigen Expression
2
Immunofluorescence Staining of Neural Markers
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Immunofluorescence staining was conducted as previously described (13 (link)). Staining was conducted overnight at 4°C with anti-NeuN (1:500; ab104225; Abcam, Waltham, MA), rabbit anti-CD68 (1:500; ab125212; Abcam), rat anti-BrdU (1:500; ab6326; Abcam) and/or rabbit anti-Iba1 (1:500; ab178847; Abcam) primary antibodies. Sections were then incubated with appropriate secondary antibodies for 60 mins at room temperature (1:500; Alexa Fluor goat anti-rabbit 594, A11037; Alexa Fluor goat anti-rabbit 488, A11008; Alexa Fluor goat anti-rat 594, A11007; Invitrogen, Waltham, MA) before mounting with DAPI Fluoromount-G (0100–20; SouthernBiotech, Birmingham, AL). Fluorescent images were captured with either an EVOS fluorescent microscope (ThermoFisher Scientific, Waltham, MA) or BZ-X800E fluorescent microscope (Keyence, Itasca, IL). Image analysis was performed using FIJI (ImageJ/Fiji, version 1.53o; National Institutes of Health, Bethesda, MD).
3
CRISPR-Mediated Gene Targeting Efficiency
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Cells grown on coverslips in six-well plates (5x105 cells/well) were transfected with sgRNA plasmid targeting Lamin A (pX330-LMNA-gRNA1, addgene 122507) and donor plasmid (pCR2.1 Clover-LMNA Donor, addgene 122507) with 2.4 µg and 0.6 µg of DNA, respectively, using JetOptimus (Polyplus). Single vectors were transfected as negative controls. Cells were imaged alive 96 hr post-transfection using a Keyence BZ-X800E Fluorescent Microscope or were fixed with 1% PFA/2% Sucrose for 10 min and permeabilized with PBS-0.5% Triton buffer for 5 min. Gene targeting efficiency was determined by counting with Cell Profiler (Positive nuclei pipeline, threshold 0.25 for clover channel intensity) the percentage of Clover-positive cells using DAPI (Thermo Fisher 40 X objective, fixed cells) or NucRed Live 647 ReadyProbes Reagent (Thermo Fisher 20 X objective, live cells). Four independent experiments were performed and at least 500 cells were counted in each experiment. Data was plotted and represented in Graph Pad version 9.
4
Targeted LMNA gene editing in cells
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Cells grown on coverslips in six-well plates (5x10 5 cells/well) were transfected with sgRNA plasmid targeting Lamin A (pX330-LMNA-gRNA1, addgene 122507) and donor plasmid (pCR2.1 Clover-LMNA Donor, addgene 122507) with 2.4 µg and 0.6 µg of DNA, respectively, using JetOptimus (Polyplus). Single vectors were transfected as negative controls. Cells were imaged alive 96 hours post-transfection using a Keyence BZ-X800E Fluorescent Microscope or were fixed with 1% PFA/2% Sucrose for 10 minutes and permeabilized with PBS-0.5% Triton buffer for 5 minutes. Gene targeting efficiency was determined by counting with Cell Profiler (Positive nuclei pipeline, threshold 0.25 for clover channel intensity) the percentage of Clover-positive cells using DAPI (Thermo Fisher 40X objective, fixed cells) or NucRed™ Live 647 ReadyProbes™ Reagent (Thermo Fisher 20X objective, live cells). Four independent experiments were performed and at least 500 cells were counted in each experiment. Data was plotted and represented in Graph Pad version 9.
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