For colocalization studies, GFP- and mScarlet-tagged NTCP constructs were transiently transfected into HEK293 cells as described (Müller et al., 2018 (link)). Cells were seeded into µ-Slide chambered coverslips (Ibidi, Martiensried, Germany) and transfected with 0.25 µg of each appropriate construct pDNA using Lipofectamine 2000 (Thermo Fisher Scientific). Finally, 50 µl of the Lipofectamine-pDNA-complex were added to the cells before incubation was started at 37°C for 2 days. Staining of the nuclei was performed with Hoechst33342 (1 µg/ml, Thermo Fisher Scientific). Fluorescence microscopy was performed on a Leica DMI6000 B fluorescence microscope (Leica, Wetzlar, Germany). All images were taken and analyzed with the Leica software LAS X.
Slide chambered coverslips
Manufactured by Ibidi
Sourced in Germany
The µ-Slide chambered coverslips are a line of cell culture products designed for high-resolution microscopy. They provide a controlled environment for cell growth and observation. The chambers feature a gas-permeable membrane and are compatible with various imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Leica dmi 6000b fluorescence microscope, by Leica camera (1 mentions)
Las x, by Leica camera (1 mentions)
Draq5, by Cell Signaling Technology (1 mentions)
Phalloidin, by Thermo Fisher Scientific (1 mentions)
Streptavidin 594, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
2 protocols using slide chambered coverslips
1
Colocalization of NTCP Constructs in HEK293 Cells
2
Immunofluorescent Labeling Protocol
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For immunofluorescent labelling, cells were seeded into 8 or 4 well µ-Slide chambered coverslips (Ibidi). Following the experimental procedures, cells were fixed using 4% PFA and quenched in 50 mM NH4Cl. Cells were permeabilized with 0.5% Saponin in 2% BSA-PBS then antibodies were diluted in the same buffer and incubated with the cells overnight at 4°C. Following three washing steps with PBS, secondary antibodies were applied for 1 h together with Hoechst (Invitrogen) and other dyes such as DraQ5 (Cell Signaling Technology), Phalloidin (Invitrogen) and Streptavidin594 (Invitrogen) depending on the experiment.
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