Cells were seeded on 35 mm glass bottom plates (Cellvis). Cells were fixed with 4% paraformaldehyde for 30 min at 4°C and permeabilized during blocking in 2% BSA containing 0.3% Triton X-100 for 1 hr at room temperature. After blocking, cells were treated with primary and secondary antibodies using standard methods. The following primary antibodies were used: OCT4 (Cell Signaling Technology, Cat. 75463S), NANOG (Cell Signaling Technology, Cat. 4903S), PAX6 (Cell Signaling Technology Cat. 60433), NESTIN (STEMCELL Technologies, Cat. 60091), TUBB3 (Cell Signaling Technology, Cat. 4466S), MAP2 (Thermo Fisher Scientific, Cat. 131500), Cytochrome c (BD Pharmingen, Cat. 556433). All secondary antibodies were conjugated to Alexa fluorophore derivatives (Thermo). See detailed information about antibodies in S7 Table . Nuclei were stained with Hoechst 3342 (Thermo). Fixed and stained cells were mounted with Fluoromount-G slide mounting medium (Electron Microscopy Sciences). Images were acquired using an Andor DU-897 camera mounted on a Nikon Spinning Disk. The software used for image acquisition and producing representative images was Nikon Elements.
Fluoromount g slide mounting medium
Manufactured by Electron Microscopy Sciences
Sourced in United States
Fluoromount-G Slide Mounting Medium is a water-based, non-fluorescent, and permanent mounting medium designed for use in fluorescence microscopy. It is formulated to preserve the fluorescent signal of specimens and provides a refractive index close to that of glass coverslips.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Du 897, by Nikon (1 mentions)
Hoechst 3342, by Thermo Fisher Scientific (1 mentions)
Cytochrome c, by BD (1 mentions)
Nanog, by Cell Signaling Technology (1 mentions)
Nestin, by STEMCELL (1 mentions)
Tubb3, by Cell Signaling Technology (1 mentions)
Anti v5 tag, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342 trihydrochloride trihydrate, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Filipin, by Merck Group (1 mentions)
Ab16048, by Abcam (1 mentions)
4 protocols using fluoromount g slide mounting medium
1
Comprehensive Immunofluorescence Imaging Protocol
2
Immunostaining of Transfected Hippocampal Neurons
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Non-transfected and transfected hippocampal neurons with either wt or m plasmid were plated on poly-D-lysine coated coverslips at a density of 100/mm2 in a 6-well plate, cultured for 14 days, and fixed by 4% paraformaldehyde treatment. For immunostaining cells were permeabilized with 0.05% Triton X-100 (Sigma Aldrich, USA) for 10 min, blocked with 1% BSA for 30 min, and incubated for 1 hour at room temperature (RT) with primary antibody Anti-V5 tag diluted 1:500 in 1% BSA (Thermo Fisher, USA, R960-25). After washing, fluorophore-conjugated secondary antibody was added for 1 hour at RT (anti-mouse 1:1000 in 1% BSA) (Jackson Immunoresearch, USA, 115-035-166). Cell nuclei were labeled using Hoechst 33342, Trihydrochloride, Trihydrate at 2μg/ml (Thermo Fisher, USA, H3570). Coverslips were mounted into the slides using Fluoromount-G™ Slide Mounting Medium (Electron Microscopy Sciences, USA).
3
Cholesterol-Induced Mitochondrial Imaging
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Huh7 cells were seeded on coverslips in DMEM (#11995-065, Gibco) supplemented with 10% of Lipoprotein Depleted Fetal Bovine Serum (#880100-5, Kalen Biomedical, LLC) overnight before treatment with 100 μM cholesterol and 10 μg/ml 58035 for 24 h. Cells were then washed with PBS and incubated with phenol red-free DMEM (#31053-028, Gibco) containing 100 nM MitoTracker Green FM (#M7514, ThermoFisher) for 30 min to allow for the internalization of MitoTracker into cells. Cells were washed with PBS three times and mounted with Fluoromount-G Slide Mounting Medium (#17984-25, Electron Microscopy Sciences). The images were visualized and captured with fluorescent microscopy (REVOLVE, Echo).
4
Cholesterol-Induced Lipid Accumulation in Huh7 Cells
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Huh7 cells were seeded on coverslips in DMEM (#11995-065, Gibco) supplemented with 10% of Lipoprotein Depleted Fetal Bovine Serum (#880100-5, Kalen Biomedical, LLC) overnight prior to treatment with 100 μM cholesterol and 10 μg/ml 58035 for 24 h. Huh7 cells were washed with PBS and immobilized by adding 4% (wt/vol) paraformaldehyde for 10 min. Cells were then washed three times with PBS and incubated with 0.1% of Triton-X for 10 min. The cells were then washed three times again in PBS and blocked with PBS containing 5% of donkey serum and 0.5% BSA for 30 min. After washing three times with PBS , cells were incubated with 0.05 mg/ml Filipin (#F4767-1MG, Millipore Sigma) and primary antibody against LaminB1 (#ab16048, abcam) at a dilution of 1:400 at a room temperature for 2 h. Cells were again washed three times with PBS and incubated with Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary antibody, Alexa Fluor 488 (#A11008, ThermoFisher) for 1 h. After another three washes with PBS, the cells were mounted with Fluoromount-G Slide Mounting Medium (#17984-25, Electron Microscopy Sciences). The images were visualized and captured with fluorescent microscopy (REVOLVE, Echo).
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