Ab9214

Immunofluorescence staining was performed on tissue and cell samples. For tissue samples, slides were deparaffinized, rehydrated, and treated with sodium citrate buffer for antigen retrieval. Slides were incubated with 3% goat serum for 30 min followed by primary antibodies. Cell samples were fixed in poly‐l‐lysine polylysine (P2100, Solarbio, China), followed by permeabilization and non‐specific binding site blocking. Samples were treated at 4°C overnight with the following primary antibodies: rabbit anti‐MPO antibody, mouse anti‐NE antibody, goat anti‐shp2 antibody (ab9214, Abcam, USA), rabbit anti‐pad4 antibody (ab214810, Abcam, USA), and rabbit anti‐histone H3 (citrulline R2) antibody (ab174992, Abcam, USA). The next day, Samples were incubated with an Alexa fluorescein‐labeled secondary antibody for 2 h. DAPI (C1002, Beyotime, China) was used to stain cell nuclei. Then, samples were imaged by an inverted confocal microscope (LSM880 with airyscan, Carl Zeiss, Germany).

The DuoLink in situ kit (Sigma-Aldrich) was used to evaluate the co-localization of SLAMF8 with SHP-2 or ALK according to the manufacturer’s instructions.^{10 (link)} We used an anti-SHP-2 antibody (goat polyclonal antibody, ab9214; Abcam, Cambridge, UK), an anti-ALK antibody (5A4), and an anti-SLAMF8 antibody (rabbit polyclonal antibody, bs-2473R; Bioss). We collected and centrifuged untreated Karpas299, untreated SU-DHL-1, untreated K562, DMSO-treated (24 h) Karpas299, DMSO-treated (24 h) SU-DHL-1, crizotinib-treated (24 h) Karpas299, and crizotinib-treated (24 h) SU-DHL-1 cells and resuspended the cell pellets in 20% (v/v) buffered formalin (pH 7.0) followed by centrifugation for 5 min at 1,500 rpm. Then the cell pellets were dehydrated and embedded in paraffin and the paraffin blocks were cut into 3–4 μm thick sections prior to hematoxylin and eosin (H&E) staining and DuoLink evaluation. The slides were mounted with fluoroshield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI). Sections were imaged with a BX63 microscope (Olympus, Tokyo, Japan) equipped with an ORCA Flash 2.8 digital camera (Hamamatsu Photonics, Shizuoka, Japan).