Ab134128

Melanoma samples and matched nontumorous tissue were obtained from the First Hospital of China Medical University. Tissues were fixed in 10% formalin, embedded in paraffin, and processed as 4-µm continuous sections. IHC staining was performed according to the manufacturers’ instructions (UltraSensitiveTM SP; MXB, China). The antibodies used included: SEMA4D (1:200; ab134128; Abcam), IFITM1 (1:2000; ab233545; Abcam), KIF20A (1:100; ab70791; Abcam) and GPR87 (1:100; ab188901; Abcam). Each sample was independently assessed by two pathologists and scored using a semiquantitative scoring system with histoscores ranging from 0 (minimum) to 300 (maximum).
This, as well as any other portions of this study involving human tissue samples, were approved by the Ethics Committee of the First Hospital of China Medical University.

IHC assays were performed using paraffin-embedded COAD and matched normal tissues. An IHC kit (Zsgb Bio, Beijing, China) was used for IHC staining. Briefly, sections were stained with 3,3-diaminobenzidine tetrahydrochloride (DAB) and haematoxylin reagent after sequential incubations with primary (Abcam, ab134128, China) and secondary antibodies. The amount and intensity of staining in three fields of view were evaluated using a microscope (Olympus, Tokyo, Japan) at 200× magnification to score the IHC staining. Each field of view was scored for staining intensity and percentage of positive cells, and the overall score was the product of the staining intensity score and cell positivity score (staining intensity score: 0 for no staining, 1 for pale yellow staining, 2 for pale brown staining, and 3 for brown staining; cell positivity score: 0 to 5% was assigned 0 points, 6% to 25% was assigned 1 point, 26% to 50% was assigned 2 points, 51% to 75% was assigned 3 points, and >75% was assigned 4 points).