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. 2013 Nov 14;8(11):e80292. doi: 10.1371/journal.pone.0080292

Figure 1. An optimized IHC assay sensitively detects VEGFR2 protein levels manipulated in vitro.

Figure 1

siRNA harboring VEGFR2 sequences were used to manipulate VEGFR2 protein levels in H441 cells to inform IHC assay development. Orthogonal techniques were used to detect VEGFR2. VEGFR2 mRNA abundance was reduced 77.7% as detected by qRT-PCR analysis (A). Protein levels for VEGFR2 were determined by three methods: western blot analysis (B), IA-MS analysis (C), and positive pixel counts of immunoreactivity of IHC on FFPE cells (D). VEGFR2 protein levels were reduced by 78.5% and by 32.9% for IA-MS and IHC, respectively. VEGFR1 siRNAs were included as controls in each panel. Error bars show standard deviation of three technical replicates for panels A and C, and the standard deviation of 2 histological spots of approximately 1000 cells each for panel D. Representative VEGFR2 IHC immunoreactivity patterns exhibiting membranous and cytoplasmic immunoreactivity in trypsinized, processed, and sectioned FFPE cells (E). Original magnification, ×1000. Scale bar: 10 µm.