Detection of Receptor Activation in FFPE Specimens Figure 4. c-MET quantification using the VeraTag FFPE assay and correlation with Western blot, ELISA and IHC in human tumor specimens. A. Quantification of c-MET in NSCLC specimens using the VeraTag FFPE proximity assay. Isotype IgG antibody signal (inset) was subtracted from c-MET assay signals. B. Western blot analysis of c-MET in corresponding NSCLC tumor lysates. b-actin levels in the blot serves as a loading control. Ci. ELISA determinations of c-MET levels in corresponding NSCLC tumor lysates. Cii. Correlation of c-MET measurements in NSCLC specimens by proximity assay and ELISA. Di. c-MET detection in NSCLC by IHC. Dii. Correlation of c-MET measurements in NSCLC by FFPE VeraTag proximity assay and IHC. Histologic score (H-score) = %IHC3+63 + %IHC2+62 + %IHC1+61 + %IHC060 was calculated as described [10]. Spearmen r and p values were calculated using graphpad prism software. doi:10.1371/journal.pone.0015932.g004 human HGF neutralizing antibody (R&D systems, Minneapolis, MN), Phospho c-Met (Tyr 1003) (13D11) (Cell signaling, Beverly, MA), Phospho c-Met (Tyr1234/1235) (D26) (Cell signaling, Beverly, MA), Phospho c-Met (Tyr1349) (Cell signaling, Beverly, MA), Rabbit monoclonal (DA1E) IgG (Cell signaling, Beverly, MA), Horseradish peroxidase (HRP) conjugated secondary antibodies (Pierce Biotechnology, Rockford, IL). Frozen or formalin-fixed paraffin embedded human tumor blocks were obtained from Asterand (Detroit, MI). Head and Neck tumors were macrodissected to remove non-tumor related content. Human Non-Small Cell lung carcinoma and human Gastric carcinoma tissues used in the study had greater than 70% tumor content. Cell lines were fixed in 10% neutral buffered formalin for 1 h at 4uC (Sigma, St. Louis, MO) and formalin-fixed paraffin embedded (FFPE) pellets were prepared by as described [10]. VeraTag reporter molecules were synthesized as described earlier [US Patent 7,105,308]. VeraTag reporter and biotin conjugated antibodies were prepared as before [10]. c-MET assay for formalin-fixed paraffin embedded (FFPE) cell lines and human tumor tissues Sections were deparaffinized/rehydrated in xylene (265 minutes), 100% ethanol (265 minutes), 70% ethanol (265 minutes), and deionized water (265 minutes). Antigen retrieval was performed with DIVA decloaker antigen retrieval buffer (Biocare medical, Concord, CA) in a decloaking chamber (Biocare medical, Concord, CA). After retrieval, slides were cooled for 1 h at room temperature, washed in deionized water, and blocked with sample blocking buffer (10% goat serum, 1.5% BSA, 1x PBS) for 1 h at room temperature. After removal of blocking buffer, sections were incubated at 4uC overnight with fluorescein-reporter conjugated and Biotin conjugated c-Met antibodies prepared in blocking buffer. Anti-c-Met (CVD13) (Invitrogen, Carlsbad, CA) was used for VeraTag Pro11 reporter conjugation; Anti-c-Met (clone 3D4) (Invitrogen, Carlsbad, CA) was used for biotin conjugation). Fluorescein-reporter reporter conjugated c-Met antibody: 0.5 mg/ml; biotin conjugated c-Met antibody: 1 mg/ ml. Isotype controls were performed as above except primary antibodies were replaced with the following antibodies: fluoresce- in-reporter conjugated c-Met antibody (CVD13): 0.5 mg/ml; Biotin conjugated IgG2a (1 mg/ml). Antibody mixture was removed by aspiration, sections were washed sequentially with buffer 1 (0.25% TX-100, 1x PBS) and buffer 2 (1x PBS). Samples were incubated for 1 h at room temperature with streptavidin- conjugated methylene blue (2.5 mg/ml) prepared in 1x PBS. Slides were washed with buffer 1 (0.25% TX-100, 1x PBS) (165 minutes) followed by 3 changes of deionized water. An illumination buffer (IB) (0.002x PBS) containing fluorescein (0.003pmol/L) and two CE internal markers was added on top of the section and samples were illuminated at 4uC for 1 h with in-house LED array that mediated photo-activated cleavage of fluorescein-reporter mole- cules. After illumination, buffer containing released reporters was collected, and run on a capillary gel electrophoresis system (ABI3100) (Applied Biosystems) under 6 kv for 50 s injection conditions for separation and detection of VeraTag reporters. Quantification of VeraTag reporter peaks was done using customized VeraTag quantitative software that normalized the peak area to the internal standard fluorescein, resulting in relative peak area (RPA) that is proportional to the concentration of the detected analyte. Tumor section area was measured using image J as described before [10]. The final value of VeraTag fluorescein- reporter was calculated by normalizing with tumor area (TA) and volume of the illumination buffer (IB) using formula RPA*IB/TA as described [10–12]. HGF assay for FFPE cell lines and human tumor tissues Antibody proximity assays were performed as described above except anti-c-Met antibodies were replaced with anti-human HGF antibodies. Following antibodies were used for the assay: Anti- human HGF (R&D systems, Minneapolis, MN) was used for VeraTag reporter conjugation, Anti-human HGF (clone SBF5) (Thermo Scientific, Fremont, CA) was used for biotin conjugation. Pro11 fluorescein-reporter conjugated Anti-human HGF anti- body: 0.5 mg/ml; biotin conjugated Anti-human HGF antibody: 1 mg/ml. Isotype controls were performed as above except primary antibodies were replaced with the following antibodies: Pro11 fluorescein-reporter conjugated Anti-human HGF antibody (0.5 mg/ml); Biotin conjugated IgG1 (1 mg/ml). HGF/c-MET ligand-receptor assay for FFPE cell lines and human tumor tissues Sections were deparaffinized/rehydrated, antigen retrieved, and blocked using a blocking buffer as described above. Samples were incubated overnight at 4uC with a mixture of unlabeled c-MET antibody (SP44) (1 mg/ml) and Pro11 fluorescein-reporter conju- gated anti-human HGF antibody (0.5 mg/ml) prepared in blocking buffer. Antibody mixture was removed by aspiration, sections were washed sequentially with buffer 1 (0.25% TX-100, 1x PBS) and buffer 2 (1x PBS). Samples were incubated with biotin conjugated goat anti-rabbit antibody (1 mg/ml) for 1 h at room temperature. After this step, samples were washed sequentially with buffer 1 and 2, incubated with nutravidin-methylene blue (0.5 mg/ml), and illuminated as described above. Fluorescein-reporter peaks were normalized with tumor area as described previously [10–12]. Isotype control assay was performed as above except primary antibodies were replaced with the following antibodies: Rabbit monoclonal (DA1E) IgG (1 mg/ml); Pro11 fluorescein-reporter anti-human HGF antibody (0.5 mg/ml). Tumors exhibited signals at least 2-fold greater than the isotype assay control were scored positive for HGF/c-MET complexes. Immunoprecipitation and Western blotting Cells were seeded in tissue culture dishes and incubated at 37uC until 60–70% confluence. Cells were starved overnight in serum free medium and stimulated with various doses of HGF (PeproTechUS, Rockey Hill, NJ) at 37uC for 10 minutes. Cells were washed with cold PBS and lysed in buffer L (50 mM Tris-HCl (pH 7.5), 1% TX-100, 150 mM NaCl, 50 mM b-glycerophosphonate, 50 mM NaF, 1 mM Na3VO4 and a cocktail of protease inhibitors (Roche)). PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e15932