SLC22a1 mutant imaging
Description
Immunofluorescent images for OCT1 mutants validated from mutational scanning experiments. Mutants were used to test the effects of variants on localization. For immunostaining experiments, HEK293 stable cell lines were plated on poly-D-lysine treated 12-well plate with sterile coverslips at a density of 200,000 cells/well and stained 2 days post-seeding when cells reached 90-100% confluency. On day of staining, cell media was removed and cells were washed with cold Hank’s Balanced Salt Solution (HBSS, Thermo Fisher Scientific Inc.). The plasma membrane was stained first using Wheat Germ Agglutin (WGA) Alexa Fluor 647 conjugate (Invitrogen Life Sciences Corporation) for 15 mins at RT, diluted in HBSS 1:500. After stain, solution is removed and cells are washed 3x with HBSS. Cells are fixed with 3.7% formaldehyde in HBSS, aspirated after 20 mins, and cells are washed again 3x with HBSS. Finally, the nucleus is stained with Hoechst solution (Thermo Fisher Scientific Inc.), diluted at 1:2000 in HBSS, for 20 mins at RT and kept in darkness. The solution is aspirated and cells are washed a final 2x with HBSS. Coverslips are carefully mounted on Superfrost Plus Microscope Slides (Thermo Fisher Scientific Inc.) with a drop of SlowFade Gold Antifade mountant (Thermo Fisher Scientific Inc.). Slides are left to dry in darkness overnight and then imaged on an inverted Nikon Ti microscope equipped with a CSU-22 spinning disk confocal. All images were captured with the following channel settings; DAPI at 300ms exposure time and 50% laser power, FITC at 300ms exposure time and 25% laser power, and CY5 at 100ms exposure time and 5% laser power. Images were overlapped using Fiji software (Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682.)