Ep1454y

Freshly cut 4-μm sections of each paraffin block of 22 ESCC samples and 19 NTST were used to perform immunohistochemistry using primary antibody against MET (EP1454Y, Abcam^®, USA). Antigen retrieval was performed in a water bath while slides were submerged in citrate buffer, pH 6.0, for 40 min at 98°C. The detection was performed following the supplier's recommendations for Novolink™ Max Polymer Detection System (Leica, UK). Samples from lung adenocarcinoma were used as a positive control of MET expression. In the negative control, the primary antibody was replaced with the antibody diluent solution.
Digital images were captured using the Aperio ScanScope CS Slide Scanner (Aperio Technologies, USA) under 20× objective magnification (0.5 μm resolution). An expert pathologist selected the tumor areas using ImageScope software suite (Aperio Technologies). The digital image analysis was performed on whole slide images with Aperio Membrane V9 algorithms (Aperio Technologies). The quantification was done automatically, after algorithm calibration by an experienced observer, and results are reported for MET immunohistochemistry (IHC) as scores from 0 to 3+, and positive tumor cell was defined at the membrane completeness between scores 1 to 3.

Whole cells were washed with cold PBS and lysed in RIPA buffer. Equal amounts of total protein (30 μg) from cell lysates were loaded onto 10% SDS/PAGE gels, transferred to PVDF membranes (Millipore), and detected using an ECL Western Blotting Detection System (Bio-Rad). The antibodies used were c-Met (1:2000 dilution, EP1454Y, Abcam) and GAPDH (1:5000 dilution, Proteintech).

HepG2 cells were plated in a cell view cell culture dish (Greiner Bio-One North America, Inc., Monroe, NC, USA) at a density of 2.5 × 10⁴ cells/compartment and incubated for 24 h. Next, the cells were exposed to NPs at a concentration of 50 μg ml^–1 and incubated for an additional 24 h, after which the cells were washed twice to remove excess NPs and then fixed with 4% paraformaldehyde (PFA) for 10 min. Subsequently, the cells were blocked with 1% bovine serum albumin (BSA)/10% normal goat serum/0.3 M glycine in PBS for 1 h, followed by washing three times (5 min each). Immediately after washing, the cells were incubated with anti–Met hepatocyte growth factor receptor (HGFR) antibody (1/100 dilution, EP1454Y, Abcam, Cambridge, MA, USA) for 1 h at room temperature (RT) and then goat anti-rabbit IgG H&L (1/200 dilution, DyLight® 488, ab96883, Abcam) in the dark for 1 h at RT. Both incubations were followed by washing three times (5 min each) with PBS. Confocal microscopy was performed using a confocal laser-scanning microscope (LSM510 META, Carl Zeiss Inc., Jena, Germany). All images were acquired using a 63 × 1.4 Plan-Apochromat oil immersion objective (Carl Zeiss).