Quantitative Dual Immunofluorescence Staining

Double immunofluorescence staining was done as described [59] (link). Rat-specific anti-DEspR mAb (10a3h10) and human-specific anti-DEspR mAb (7c5b2) were labeled with AlexaFluor(AF)-488 or AF568, and used at 10 ug/ml for cells or frozen sections, and at 100 ug/ml for fixed, paraffin-embedded sections following antigen-retrieval. Anti-alpha-smooth muscle actin, aSMA (SIGMA-ALDRICH, MO), anti-CD133 (Creative Biomart, NY) and human-specific anti-CD31 (Sta. Cruz Biotechnology, TX) were used per manufacturer's specifications. For quantitation of immunofluorescence results of tumor biopsy cores, digital photomicroscopy was performed using a Zeiss Axioskop fluorescence microscope using identical exposure settings for tumor sections and normal controls once ideal settings determined for positive target-specific fluorescence. For quantitation of relative fluorescence intensity levels, auto-exposure times in milliseconds were recorded using identical microscopy settings in photomicroscopy sessions.