Figure 1
From: Evaluation of stromal HGF immunoreactivity as a biomarker for melanoma response to RAF inhibitors
Validation of immunohistochemistry protocol for hepatocyte growth factor (HGF). (a) Quantitative RT-PCR analysis (top) of R2F1 fibroblasts showing mRNA for control vector (Vec) and HGF-expressing vector (HGF); western blot (bottom) reveals band from recombinant HGF (rHGF), transfected cell supernatant (HGF-trans), and vector control (Vec). (b) R2F1-transfected fibroblasts were stained for HGF applying the immunostaining protocol proposed by Straussman et al2 (top row). Note strong staining in HGF-transfected cells (HGF trans), strong staining in fibroblasts transfected with control vector (Vec control) and also high background staining in the isotype-matched irrelevant control. A simplified staining protocol (bottom row) showed immunoreactivity only in HGF-transfected fibroblasts and not in vector control-transfected cells; the isotype control was negative. (c) Human normal placenta stained with Straussman et al2 method (upper panel) and with a modified protocol that eliminates the tertiary antibody and uses an alkaline phosphatase detection system with a red chromagen (lower panel); insets show cytotrophoblasts (lower third) and mesenchymal cells. (d) Human normal skin exposed to IL-1β for 48 h, stained for HGF following Straussman et al2 (upper panels). Note the similarity at × 400 between an HGF+ dermal cell (asterisk) and an elastic fiber (encircled, arrows point other fibers). Oil immersion ( × 1000) microphotograph (inset) allows distinction between the otherwise equivocal tissue components. Same sample of human skin exposed to IL-1β for 48 h stained for HGF using the modified protocol does not present elastic fiber staining that could be misinterpreted as HGF+ dermal cells (lower panels). (e) Normal skin showed lightly counterstained nuclei with minimal immunoreactivity (left column); skin cultured in media alone and with IL-1β for 48 h showed progressive increase in expression of HGF (middle and right columns), with IL-1β-treated skin containing cells that showed maximal (score 3) HGF immunoreactivity.
