Fitc conjugated secondary antibody

Tumour cells were grown to near confluence in DMEM/10% FBS in a Lab-Tek Parmanox eight-well chamber culture slide (VWR, East Grinstead, UK). The cells were incubated with anti-EGFR antibodies for 1 h on ice or 37 °C (for internalisation studies) and then fixed with 4% paraformaldehyde for 10 min at room temperature. Cell membranes were permeabilised with 0.5% Triton X-100 (Sigma-Aldrich) in PBS/1% BSA for 20 min at room temperature and blocked with PBS/3% BSA for 30 min. The tumour cells were then incubated with FITC conjugated secondary antibody (AbD Serotec, Oxford, UK) for 1 h at 4 °C. Following washes in PBS, cells were incubated with 1 μg ml⁻¹ of Hoescht 33258 nuclear stain (Sigma-Aldrich) for 5 min at room temperature and mounted with H-1000 mounting medium (Vector Laboratories, Peterborough, UK), cover slipped (VWR) and photographed using FITC filter array on a Nikon eclipse i80 fluorescent microscope.