Immunostaining was performed on a Dako Autostainer per manufacturer’s instructions (CSA kit, Agilent Dako) 84 (link),85 (link). Prior to immunostaining, RPPA slides were incubated in 1X ReBlot Mild solution (Fisher Scientific), washed twice with phosphate buffered saline without calcium or magnesium (PBS), and incubated in I-Block solution (Invitrogen T2015) at room temperature for a minimum of 30 min. Each slide was incubated with a single primary antibody at room temperature for 30 min. Antibody specificity was confirmed by Western blotting as previously described 86 (link). The negative control slide was incubated with antibody diluent. Secondary antibody was gt anti-rabbit IgG H+L (1:10,000) (Vector Labs, Burlingame, CA) or anti-mouse IgG (1:10, Agilent Dako CSA kit). Signal detection was amplified via horseradish peroxidase mediated biotinyl tyramide deposition with chromogenic detection (Diaminobenzidine) per manufacturer’s instructions (Agilent Dako).
Arrays were scanned at 600dpi, grayscale, on a flatbed scanner (UMAX PowerLook). Spot (pixel) intensity was analyzed using ImageQuant ver 5.2 (GE Healthcare), with mean local area background subtraction 80 (link). Signal:background intensity for each spot was calculated using the freely available data reduction algorithm (RAS ver16,www.capmm.gmu.edu ) 83 (link). Spot intensities were normalized to total protein/spot.
Arrays were scanned at 600dpi, grayscale, on a flatbed scanner (UMAX PowerLook). Spot (pixel) intensity was analyzed using ImageQuant ver 5.2 (GE Healthcare), with mean local area background subtraction 80 (link). Signal:background intensity for each spot was calculated using the freely available data reduction algorithm (RAS ver16,