Alexa fluor 488 goat anti rabbit igg

Confocal microscopy was carried out as described previously (13 (link), 25 (link), 30 (link)). Briefly, HEK293 cells seeded onto the coverslips (1.0 × 10⁵ cells/ml) were transfected with the WT or mutant LDLR cDNA as indicated. At 48 h later, cells were fixed with 4% paraformaldehyde and permeabilized with cold methanol. After blocking with 1% BSA, the cells were incubated with an anti-LDLR monoclonal Ab and an anti-Na⁺/K⁺-ATPase polyclonal Ab (1:200) (Abcam). Ab binding was detected using Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 568 goat anti-mouse IgG. After washing, coverslips were mounted on the slides with Antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Localizations of LDLR and Na⁺/K⁺-ATPase in the transfected cells were determined using a Leica SP5 laser-scanning confocal microscope (filters: 461 nm for DAPI, 519 nm for Fluor 488, and 603 nm for Fluor 568).