Fluoview fv500 laser confocal microscope

A549, H1299, and PKR−/− MEF cells (5 × 10⁴ cells/well) were grown on chamber slides until 70% confluence and then treated with PBS, Ad-luc, Ad-PKR, Ad-PKRΔ6, Nu-PKR, Nu-PKRΔ6, or radiation. Fourty-eight hours later, cells were washed with PBS and fixed with 4% paraformaldehyde/PBS for confocal analysis, as previously described [10 (link), 28 (link)]. Cells were blocked with 1% normal goat serum for 1 hour and then incubated overnight at a dilution of 1:100 with the primary anti-PKR antibody. To remove the primary antibody, the slides were rinsed with PBS and incubated with a fluorescein isothiocyanate- or rhodamine-conjugated secondary antibody (Invitrogen) for 1 hour. The slides were then mounted with the ProLong Gold antifade reagent containing 4′, 6-diamidino-2-phenylindole (DAPI; Invitrogen) and analyzed under an Olympus FluoView FV500 laser confocal microscope (Olympus America, Melville, NY) after adjustment for background staining.

Cells were seeded on glass coverslips in 12-well plate at a density of 1 × 10⁵ cells/well. After 2-day culture, cells were fixed in cold methanol, permeabilized in buffer (0.1% BSA, 0.3% Triton X-100 in PBS), and blocked with goat serum dilution buffer (10% goat serum, 1% Triton X-100, 10 mM glycine in PBS, GSDB). Slides were incubated with LAT1 (1:100, Trans Genic Inc., Fukuoka, Japan) or 4F2hc (1:100, Santa Cruz Biotechnology, Dallas, TX, USA) diluted in GSDB buffer overnight. Cells were incubated with Alexa Fluor 594 conjugated anti-mouse IgG (1:100 Life Technologies, Carlsbad, CA, USA) and DAPI (1:500; Roche, Basel, Switzerland) diluted in GSDB buffer, and then mounted in fluoro-KEEPER Antifade Reagent (nacalai tesque, Kyoto, Japan). Fluoro-images were captured by Fluoview FV500 Laser confocal microscope (Olympus, Tokyo, Japan).