Fluorescent anti mouse secondary antibody

Human and mouse tumours were stained with anti-COX2 rabbit monoclonal antibody (Cell Signaling Technology, 12282), anti-S100 rabbit polyclonal antibody (Dako, Z0311, used to identify nerves on paraffin-embedded tissue), and cytokeratin AE1/AE3 mouse monoclonal antibody (Pan cytokeratins, EMD Millipore, IHCR2025-6, used to identify tumour cells on paraffin-embedded tissue). Mouse IgG (Dako, X0931) or rabbit IgG (Dako, X0936) was used at the same concentrations as the primary antibodies as a negative control. Biotinylated goat anti-mouse or anti-rabbit secondary antibodies were used (Biocare Medical, GM601 and GR608). Hemotoxylin and eosin staining was performed to assess tumour morphology. Immunofluorescence on frozen CAM sections was performed using human collagen IV antibody^{64 (link)} (1:1000) followed by incubation with DAPI. Immunofluorescence labelling of neurites was performed as follows: nerve explants were blocked with goat serum and incubated with anti-neurofilament (NF-M 160kD chain primary antibody; Zymed Laboratories, 13-0700; 1:500) in 0.3% triton X-100, followed by incubation with fluorescent anti-mouse secondary antibody (Jackson ImmunoResearch, 115-006-075). Imaging of representative fields was performed using an Olympus BX-51 microscope.