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Labeling of PM and ER with protein tags

LC Ling Chu
JT Jonathan Tyson
JS Juliana E. Shaw
FR Felix Rivera-Molina
AK Anthony J. Koleske
AS Alanna Schepartz
DT Derek K. Toomre
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Generation of hTERT RPE-1 cells stably expressing Smo-Halo was described previously using pLVX-ss-HaloTag-Smo24. hTERT RPE-1 cells stably expressing Smo-Halo were transfected with Sec61β-SNAP using FuGENE HD Transfection Reagent (Promega) one day before imaging. Before imaging, the cells were incubated with 500 μL of 2 μm SiR-CA and 2 μm Yale595-BG in Live Cell Imaging Buffer for 30 min at 37 °C. After washing, STED imaging was carried out at 37 °C using DMEM as the imaging buffer under Leica SP8 microscope with two excitation lasers at 595 nm (10% power), 650 nm (60% power) and two detection windows at 605–625 nm and 670–750 nm. The 775 nm depletion laser was used for STED microscopy (30% power). Images were recorded continuously at 2 s per frame. Raw image data were spectral unmixed Gaussian blurred (2.0 pixels) in ImageJ.

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