Phycoerythrin conjugated secondary antibody

HeLa cells were seeded (5 × 10³ cells/well) into six-well cell culture dishes containing sterile coverslips, beadfected, and incubated for 48 h at 37 °C with 5% CO₂. The coverslips were then washed with PBS (three 2-ml washes; Invitrogen, Paisley, UK) and fixed with 1% paraformaldehyde (20 min, 4 °C). Coverslips were again washed (three washes, each with 2 ml of PBS), permeabilized with 1% Trition X-100 in PBS for 30 min at room temperature (permeabilized samples only), and incubated with anti-GFP monoclonal antibody (Chemicon, Watford, UK) at 1:1000 dilution in PBS containing 4% w/v BSA with gentle rocking for 2 h at room temperature. Coverslips were washed as before and incubated with phycoerythrin-conjugated secondary antibody (Sigma, Poole, UK) (1:500 dilution in PBS containing 4% BSA) for 1 h with gentle rocking. Coverslips were washed and dried before being mounted in mounting medium (Vector Laboratories, Peterborough, UK). Slides were imaged with a Zeiss LSM 510 Meta confocal microscope using a Plan Apochromat ×63/1.40 objective mounted on an Axioplan 2 motorized upright stand, with images collected by LSM software (Zeiss). GFP fluorescence was detected using a Zeiss bandpass (505–550 nm) filter after excitation at 488 nm. Phycoerythrin fluorescence was detected using a long-pass 560-nm filter after excitation at 543 nm.