47 MET immunoreactivity and poor prognosis PBS and immersed in fresh, cold Paraformaldehyde (4% v/w) for 20 min. for fixation. After blocking endogenous peroxidase activity with a 0.3% H2O2 solution for 5 minutes, slides were stained with the ABC procedure (40). Endogenous biotin was blocked using the Avidin/Biotin blocking kit (SP-2001; Vector Laboratories Ltd; Peterborough, United Kingdom). Cells were incubated O/N at 4°C with the primary MET antibodies (Supplementary Table S3) and incubated for 30 minutes at room temperature with the secondary biotinylated polyclonal swine anti-rabbit antibody (1:150; E0431; Dako; Heverlee, Belgium). Signal amplification was performed with the VECTASTAIN® Elite ABC system (PK-6100; Vector Laboratories Ltd) according to the manufacturer’s protocol. Finally, horseradish peroxidase activity was visualized in 90 seconds with 3, 3’-diaminobenzidine (K3468; Dako) prepared in accordance with the manufacturer’s instructions. Formalin fixation and paraffin embedding of MET antibody cell line panel Cells were cultured in triplicate in T175 flasks until 75-90% confluence was reached. Subsequently, per cell line, all three cultures were harvested in a single volume of PBS (10 mL). After removing the PBS, the cells were fixed O/N at 4°C with 10% formalin (10 mL). After removing the fixative, the cells were resuspended in PBS (500 mL) to be transferred to a flat bottom embedding capsule (#70021; Electron Microscopy Sciences; Hatfield, PA, USA). Herein, after centrifugation and removal of the supernatant, the cells were dissolved in a PBS-agar (5.0%; Life Technologies™/ Invitrogen™) solution (500 mL) of 56°C. Upon solidification on ice, the cell containing agar blocks were removed from their embedding capsules, transferred to cassettes and embedded in paraffin. Immunohistochemical staining of FFPE MET antibody validation cell line panel, whole tissue sections and TMA sections After deparaffinization and rehydration, endogenous peroxidase activity was inactivated by incubating the sections in 3% H2O2 for 10 min. Subsequently, antigen retrieval was carried out by heating the sections under high pressure – up to 0.9 bar in case of the FFPE cell lines and up to 1.2 bar in case of the FFPE tissues – in Tris-EGTA buffer (0.01 M Tris, 0.001 M EGTA, pH 9.0). After antigen retrieval, the tissue sections were stained with the ABC procedure using an almost identical protocol applied for the immunochemical staining of the MET antibody validation cell line panel. However, in contrast to the immunocytochemical procedure, the sections were incubated with 0.22% bovine serum albumin solution (A7034; Sigma-Aldrich®) in 1X PBS for 7 min. after blocking endogenous 3
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