Ab1377

To begin, the tissue sections were dewaxed and hydrated. Then the antigens were retrieved in a citrate repair solution. The tissue sections were subjected to blocking in Tris buffered saline solution containing 10% normal serum and 1% bovine serum albumin for 2 h at room temperature. Each section was then treated with EDD antibody (1 : 100, PA5-34430, Invitrogen), CDCP1 (1 : 100, ab1377, Abcam), and METTL3 (1 : 500, ab195352, Abcam) at 4° C overnight. Normal serum was used as the NC instead of using a primary antibody. Then, 50 μL of 3% H₂O₂ was added to each section, which was followed by incubation at room temperature for 20 min with the purpose of eliminating endogenous peroxidase activity. Next, 50 μL aliquot of polymer enhancer was added to each section, which was followed by incubation of the sections at 37° C for 20 min. Secondary antibody (50 μL, Abcam, ab205718, goat anti-rabbit, 1 : 2000) was then added dropwise to each section, and incubated at 37° C for 30 min. The sections were exposed to diaminobenzidine reagent, and hematoxylin counterstaining. The brown color represented positive expression.

Paraffin sections were deparaffinized and rehydrated. Heat-induced antigen retrieval was carried out for 15 min in citrate buffer. After endogenous peroxidase was blocked with 3% hydrogen peroxide and nonspecific antigens were blocked with 5% bovine serum albumin, incubation was performed with antibodies against CDCP1 (Abcam, catalog #ab1377, human, 1:100), CD44 (Abcam, catalog #ab189524, human, 1:100), and ITGAM (Cell Signaling Technology, catalog #23743, human, 1:100). The next day, the secondary antibody was added after washing with PBS three times. Subsequently, sections were counterstained with hematoxylin before examination by microscopy.

Total protein in tissues or cells was extracted with a Radio Immunoprecipitation Assay lysis buffer solution containing phenylmethyl sulfonylfluoride. Cells were then centrifuged at a temperature of 4° C and at the rate of 8000 ×g for the duration of 10 minutes to harvest the supernatant. The bicinchonininc acid kit (P0012, Beyotime Institute of Biotechnology, China) was employed to measure the total protein concentration. Then, 50 μg of protein was dissolved in 2 × sodium dodecyl sulfate (SDS) loading buffer. After boiling both at 100° C for 10 min, each sample was subjected to SDS-polyacrylamide gel electrophoresis. The protein was transferred to a polyvinylidene fluoride (PVDF) membrane using wet transfer. After blocking the PVDF membrane for 1 h, it was incubated at 4° C overnight with diluted primary antibodies: rabbit anti-EED (1 : 1000, PA5-34430, Invitrogen), rabbit anti-METTL3 (1 : 1000, ab195352, Abcam, Cambridge, UK), rabbit anti-CDCP1 (1 : 1000, ab1377, Abcam), and murine anti-β-Actin (1 : 5000, ab8227, Abcam). This was followed by incubation with secondary antibody to immunoglobulin G (IgG) (Abcam, ab205718, goat anti-rabbit, 1 : 20000; Abcam, ab205719, goat anti-mouse, 1 : 20000) at room temperature for 1 h. The blots were developed with the enhanced chemiluminescence substrate (WBKLS0100, Millipore, Billerica, MA, USA) and quantified using the Image J software.