Q dot linked fluorescent secondary antibodies

An EGFRvIII-expressing human GBM (GBM39) was propagated as a xenograft^{40 (link)} and kindly provided by C. David James (Department of Neurological Surgery, University of California San Francisco). Harvested tumor tissue was formalin-fixed, paraffin-embedded, and cut into 4 µm sections for mounting on positively-charged slides. Antigen retrieval was performed using protease 2 (Ventana). Sections were immunostained with primary antibodies targeting phospho-Tyr-845 (1:150; Abcam) and human uPAR (1:75; Dako) for 1 h at 37°C using the Ventana Discovery Ultra Platform. Q-dot-linked fluorescent secondary antibodies (1:150; Invitrogen) were added for 1 h. The slides were rinsed and cover-slipped with Prolong Gold and DAPI (Invitrogen). Slides were visualized on a Zeiss Axio Imager2 using Cambridge Research Instruments Nuance Multispectral Imaging System software to capture images and visualize individual fluorophore spectra free from auto-fluorescence noise. In control experiments, phospho-epitope labeling was validated using protein phosphatase treatment, which eliminated signal.