Ab233545

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.

Isolated leukocyte and lymphocyte samples were fixed in 4% paraformaldahyde in PBS (0.2 mM and pH 7.4) and spun in a cytocentrifuge (8 min at 300g) onto coated slides. Slides were permeabilized and blocked with 10% normal donkey serum in PBS (with 0.5% Triton X-100), and primary antibodies (S100A8/9, Abcam, ab22506; IFITM1, Abcam, ab233545) were incubated at 4 °C overnight, followed by incubation with donkey anti-rabbit Alexa Fluor 488 (Invitrogen, A32790) or anti-mouse Alexa Fluor 555 (Invitrogen, A31570) for 1 h at room temperature. Slides were sequentially stained with CD24 (Abcam, ab202073) on the same slides for 1 h at room temperature, followed by donkey anti-rabbit Alexa Fluor 647 (Invitrogen, A31573). Imaging was done using a VS-120 slide scanner (Olympus), and high-resolution imaging was done using an SP8 spectral confocal microscope (Leica). Image processing was completed in Fiji (version 2.1.0)^{60 (link)}.