Detection of Receptor Activation in FFPE Specimens Figure 5. VeraTag FFPE quantification of HGF and correlation with Western blot, ELISA and IHC in cell lines. A. VeraTag FFPE quantification of HGF in unstimulated and HGF stimulated A549 cells. Isotype IgG control signals (inset) were subtracted from the HGF signals. Bi. VeraTag quantification of HGF in Ln18, Ln229, U138, U118, and U87MG glioma cell lines. Isotype IgG control signals (inset) were subtracted from HGF assay signals. ND: Not detectable. Bii. ELISA determinations of HGF in A549, Ln18, Ln229, U138, U118, and U87MG cell lysates. ND: Not detectable. Ci. VeraTag FFPE quantification of HGF in HEK293/HGF (clone 1) and U138 glioma cell lines. Cii. IHC detection of HGF in HEK293/HGF stable clone (clone 1) and U138 glioma cells. doi:10.1371/journal.pone.0015932.g005 Lysates were incubated for 10 minutes on ice, centrifuged at 13,000 rpm at 4uC for 10 minutes, and clear supernatant was collected. Human tumor tissue lysates were prepared by first adding frozen tumors in liquid nitrogen and then grinding tumors using a pestle. Following sufficient grinding and mixing, cold buffer L was added, samples were incubated for 20 minutes, centrifuged at 13000 rpm, and clear supernatant was collected. Protein concentration was measured using bicinchoninic acid reagent (Pierce Biotechnology, Rockford, IL). Total cell lysates were run on SDS-PAGE and western blots were probed with various antibodies. Primary antibodies used for probing immuno- blot are as follows: Anti-c-Met (clone 3D4) (Invitrogen, Carlsbad, CA), Anti-human HGF (clone SBF5) (Thermo Scientific, Fremont, CA), anti-b actin (Clone AC-74) (Sigma, St Louis, MO). Immunoprecipitations were carried out using standard procedures and captured on protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA). We used 200 mg and 2 mg of total protein, respectively, for cell line and tumor lysates. Primary antibodies used for immunoprecipitation are as follows: Anti-c-Met (clone 3D4) (Invitrogen, Carlsbad, CA), Anti-human HGF (clone A10) (Enzo Life Sciences, Plymouth meeting, PA) Peptide ELISA A series of 43 peptides that span the entire intracellular domain of c-MET (K955-S1390) were custom ordered (Elim biopharma- ceuticals, Hayward, CA). Each peptide was 14–15 amino acids long and contained 5 amino-acid overlapping sequence with the adjacent peptide. Peptides were absorbed on the ELISA plates overnight at 4uC. Plates were blocked with 3% BSA in PBS containing 0.05% Tween 20 (PBSTw). Following 2 PBSTw washes, antibodies were added. Following 3 washes, bound antibody was detected with Horseradish peroxidase labeled secondary antibody. c-MET and HGF ELISA Total c-MET ELISA assay was performed as per manufactur- er’s instructions using a kit (R&D systems, Minneapolis, MN). Total HGF ELISA assay was performed as per manufacturer’s instructions using a kit (RayBiotech, Norcross, GA). Immunohistochemistry and H&E staining Samples were deparaffinized/rehydrated and heat-retrieved as described above. Sections were blocked with blocking buffer (10% BSA, 1.5% BSA, 1 mg/ml human IgG, 1x PBS) for 1 h at room temperature and then incubated with either anti-c-Met (CVD13) (Invitrogen, Carlsbad, CA) (1:400 dilution) or Anti-human HGF (clone SBF5) (Thermo Scientific, Fremont, CA) (1 mg/ml) for 1 h at room temperature. Sections were washed sequentially with wash buffer 1 (0.25% TX-100, 1x PBS) and wash buffer 2 (1x PBS) and incubated with secondary antibodies (Vector labs, Burlingame, CA). Visualization of the c-Met or HGF on the sections was done using an immunoperoxidase system (Vector labs, Burlingame, CA) as per manufacturer’s instructions. Histologic score (H-score) = %IHC3+63 + %IHC2+62 + %IHC1+61 + %IHC060 was calculated as described [10]. H&E staining of the tissues were performed using standard procedures. Cell micrographs were taken using a digital image camera mounted on a leica microscope. Covalent cross-linking and Surface Protein-Protein Interaction by Cross-linking ELISA (SPPICE) Covalent cross-linking using sulfo-EGS (Pierce Biotechnology, Rockford, IL) was done as described [13]. After cell lysis, extracted proteins were collected, and immunoprecipitated with Anti- human HGF antibody (clone A10) (Enzo Life Sciences, Plymouth meeting, PA). Eluted proteins were separated on SDS-PAGE using Tris-glycine Gels, transferred to nitrocellulose membrane, and immunoblotted with anti c-MET antibody (CVD13) (Invitrogen, Carlsbad, CA). To detect and quantify complexes by SPPICE assay, cross- linked proteins were extracted after cell lysis and added to 96 well microtiter plate pre-coated with c-MET capture antibody (R&D systems, Minneapolis, MN). After overnight incubation at 4uC, liquid was removed, followed by 3 washes with wash buffer (0.05% Tween-20, 1xPBS). A biotin conjugated Anti-human HGF detection antibody (RayBiotech, Norcross, GA) was added, followed by 3 washes with 1xPBS. Detection of the complexes was performed by adding streptavidin labeled Horseradish peroxidase (RayBiotech, Norcross, GA). Plates were developed by adding substrate as per manufacturer’s instructions (R&D systems, Minneapolis, MN). Isotype IgG control assay was performed as above except extracted protein samples were added to 96 well microplate pre-coated with mouse IgG antibody (Becton Dickenson, Franklin lakes, NJ). OD450 was calculated by subtracting isotype IgG background from the assay specific signals. Results Proximity-based assays for the detection and quantification of ligand, receptor, and ligand- receptor complexes Proximity-based VeraTag assays are antibody-based detection methods to quantify proteins and protein complexes. This approach has been successfully applied to the measurement of HER2 receptor activation by detecting and quantifying HER2 and HER2 dimer levels in clinical FFPE specimens and soluble cell lysate formats [10–12,14–15]. A schematic of the FFPE assay work-flow is shown in Fig. 1A. Briefly, after deparaffinization and antigen retrieval, tissue sections are incubated with a mixture of fluorescein-reporter antibodies plus biotin-conjugated antibodies that recognize one or more target analytes. As a second step, a streptavidin (SA) conjugated photosenstizer (methylene blue; MB) is added which binds to the biotin-conjugated antibody. In a third step, the tissue section is illuminated with red light (670 nm) which triggers the SA-MB photosensitizer to convert dissolved O2 in the buffer to reactive singlet oxygen (1O2). This reactive state of the oxygen in turn cleaves thioether bonds linking the fluorescein- reporter to conjugated reporter antibodies that are bound in close proximity to the biotin-conjugated antibodies. The proximal selectivity of the 1O2 induced thioether cleavage is approximately 30 nm to PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e15932