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Finally, cells were mounted using Vectashield fluorescent medium and visualized with fluorescence microscopy.
Cells were mounted with Vectashield fluorescent medium and viewed with a fluorescence microscope.
Cells were incubated in DMEM containing 10 μM H2-DCFDA at 37°C for 30 min. Cells were washed in PBS, mounted with Vectashield fluorescent medium, and viewed with a fluorescence microscope.
Cells were incubated in DMEM containing 10 μM H2-DCFDA at 37°C for 30 min. Cells were washed in PBS, mounted with Vectashield fluorescent medium (Burlingame, CA, USA), and viewed with a fluorescence microscope.
Consequently, fluorescent medium in ΔB and fluorescent cells of ΔC and ΔBC were observed in the microfluidic channels.
Consequently, we observed fluorescent medium in ΔB (Figure 2B, C-2) and fluorescent cells in ΔC (Figure 2B, C-3).
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The cells were washed once using 1× PBS buffer and then resuspended in non-fluorescent medium.
One way to limit the influence of autofluorescence is to dilute Vectashield in a non-fluorescent medium.
For all fusion proteins the linker sequence is as described in Gruber et al. (2006).> For microscopy, cells were resuspended in non-fluorescent medium (SD TRP) and either mounted on a glass cover slip (for still images or movies of max. 5 min duration) or immobilized on a 2% agar pad containing non-fluorescent medium.
Living cells from asynchronous cultures grown to early log phase were suspended in non-fluorescent medium, mounted on a slab of 2% agarose, and sealed beneath a coverslip with paraffin wax.
Briefly, to measure the dynamics of NPCs in cells with and without accumulated plasmids, cells were grown in SD -URA, restreaked on SD LEU containing β-estradiol (1 µM final concentration; Sigma) 16 18 hr prior to imaging, and then immobilized on a 2% agar pad containing non-fluorescent medium.
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