Hoescht 33258 nuclear stain

Immunocytochemistry was used to examine expression of the human DMT1 protein in hNPC. Cover slips were fixed with 4% paraformaldehyde (PFA) and washed with phosphate-buffered saline (PBS) before incubation in blocking buffer (PBS, 5% normal donkey serum (NDS), 0.2% Triton X-100 (Sigma-Aldrich)) for 30 minutes. The anti-DMT1 primary antibody (mouse monoclonal; Sigma-Aldrich) was diluted 1:500 in blocking buffer. Cells were incubated in primary antibody for 1 hour at room temperature then washed with PBS. Secondary antibody (donkey anti-mouse IgG Alexa Fluor 488; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was diluted 1:1,000 in blocking buffer and then applied to cells for 30 minutes. After another PBS wash, coverslips were incubated in Hoescht 33258 nuclear stain (Sigma-Aldrich) for 3 minutes before mounting coverslips on glass slides with Fluoromount (Southern Biotech, Birmingham, AL, USA). Images were collected using a Nikon Eclipse fluorescence microscope, a Nikon Intensilight camera, and NIS Element D software (Nikon, Tokyo, Japan). ImageJ software was used for image processing and cell counting. For each expression time point, at least six fields of view on each of three coverslips were analyzed for percent of cells over-expressing DMT1.

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.