Bs 1646r

The tissues to be tested were treated with xylene and ethanol for dehydration. Consequently, the samples were treated in 10 mM sodium citrate solution (pH=6.0) at 100°C for 14 minutes and cooled for 25 minutes for antigen retrieval. The endogenous peroxidase activity was eliminated using 3% hydrogen peroxide, and the internal non-specific binding sites were sealed using Tris-buffered saline with Tween 20 (TBST) containing 3% bovine serum albumin. Tissue sections were probed overnight at 4°C with monoclonal antibody to SHP2 (1:100, ab32083, Abcam, Cambridge, UK), TET3 (1:100, ab139311, Abcam), p-EGFR (1:500, ab40815, Abcam), Phospho-ERK1 (Thr183, Tyr185, 1:500, BS-1646R, Thermo Fisher Scientific Inc.) and with HRP-labeled secondary anti-immunoglobulin G (IgG; 1:1,000, ab6721, Abcam) for 35 minutes at 37°C. Next, the chromogen reaction was detected by staining the sections with diaminobenzidine and hematoxylin. Then, immunohistochemistry staining was quantified and finally scored according to the degree of staining (1 negative staining, 3 light yellow, 5 light brown, and 7 dark brown) and the range of positivity (1 score 0%-20%, 2 scores 21%-40%, 3 scores 41%-60%, 4 scores 61%-80%, and 5 scores 81%-100%).

The proteins in cells or tissues were extracted using the Whole Protein Extraction Kit (BC3711, Beijing Solarbio Life Sciences Co., Ltd., Beijing, China). The extracted proteins were mixed with the loading buffer. Equal amounts of extracted proteins were pipetted onto 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). Sealing was performed using 10% skim milk. Samples were probed with the indicated primary antibodies: SHP2 (1:5,000, ab32083, Abcam), p-EGFR (1:2,500, ab40815, Abcam), Phospho-ERK1 (Thr183, Tyr185) (1:1,000, BS-1646R, Thermo Fisher Scientific Inc.), and GAPDH (1:1,000, ab8245, Abcam) overnight at 4°C. After washing with TBST, horseradish peroxidase (HRP)-conjugated secondary antibody to IgG (1:1,000, ab6721, Abcam) was incubated with the membranes. Membranes were exposed and imaged using an ECL kit (P0018S, Beyotime Biotechnology Co., Ltd., Shanghai, China), and band intensities were quantified by ImageJ software.