Immunofluorescence Localization of Proteins

HEK-293T cells or TREx BCBL1-Rta cells expressing ORF11-FLAG were grown on sterilised glass coverslips treated with Poly-L-Lysine (Sigma-Aldrich). After 24 h cells were washed in PBS and fixed in PBS containing 4% (v/v) paraformaldehyde for 10 min, washed twice in PBS and permeabilised using PBS containing 1% Triton X-100 for 10 min^{58 (link),59 (link)}. Coverslips were then incubated with anti-FLAG or anti-LANA and Alexa Flour 546 (Invitrogen), goat anti-Rat IgG or goat anti-Rabbit IgG respectively, for 1 h each at 37 °C before being mounted onto microscope slides using Vectashield® with DAPI. Slides were visualised on a Zeiss LSM 700 laser scanning confocal microscope and images analysed using Zen® blue 2.0 (Zeiss).