8 chambered polystyrene culture treated glass slides

Immunocytochemistry was used to demonstrate the  presence of EN2 in cells. Cell lines were grown overnight in an 8-chambered polystyrene culture treated glass slides (BD Biosciences, UK), fixed with 4% paraformaldehyde (Sigma, UK) and washed with PBS three times. Next, the cells were incubated with 10% horse serum (Jackson ImmunoResearch, USA) for 20 minutes and further with anti-EN2 goat polyclonal IgG primary antibody (Abcam, UK) diluted 1:100 in PBS/1% BSA at room temperature for 2 hours or overnight at 4 °C. A ‘no primary antibody’ sample was included containing PBS/1% BSA alone for use as a negative control. After primary antibody incubation, the cells were washed three times with PBS and incubated with donkey anti-goat secondary antibody (Abcam, UK) at 1:200 in the dark for 45 minutes. After incubation, cells were washed three times with PBS, chambers removed and slides mounted using propidium iodide (PI) Vectashield® mounting medium (Vector Laboratories, USA). The slides were imaged at X40 magnification using the Nikon A1M confocal microscope and NIS elements acquisition software (Nikon, UK). Images were processed with ImageJ.

Cell lines were incubated for monolayer growth in 8-chambered polystyrene culture treated glass slides (BD Biosciences, UK) with appropriate media. Staining was compared in the absence of primary antibody, and in non-permeabilised and permeabilised cells (0.2% Triton X-100 (Sigma-Aldrich, UK). Cells were incubated in 5 μg/ml Wheat-Germ Agglutinin cell membrane stain (Invitrogen, UK) for 10 min at 37 °C, before fixation with warm 4% paraformaldehyde, and blocking with 4% horse serum. Cells were incubated overnight in polyclonal goat anti-EN2 antibody (Abcam, UK) diluted 1:100 in 1% BSA (Sigma-Aldrich, UK) in PBS or in 1% BSA in PBS alone. This primary antibody has been used within the department for immunofluorescence work in multiple cells lines derived from varying tumour types, with consistent results. The secondary antibody Alexa Fluor 488 donkey anti-goat IgG (Invitrogen, UK) diluted 1:200 in 1% BSA/PBS was added, along with the nuclear stain TO-PRO-3 (Life Technologies, UK) diluted 1:400, at room temperature for 1 h. The slides were mounted and visualised using a Zeiss LSM 510 confocal laser scanning microscope, and a Plan-Apochromat 40× oil immersion objective. Images were recorded and analysed using ZEN 2009 capture software.