Ab108393

The 24-well cell culture plates were divided into H1 and H9 groups. Cell climbing sheets were placed and coated with Matrigel working solution. The cells were rinsed twice with PBS, incubated with 4% paraformaldehyde on ice for 15 min, rinsed three times with pre-cooled PBS, added with 1 mL blocking solution (P0260, Beyotime, China) and incubated for 30 min, incubated with antibody MIF (1:800, GTX53741, GeneTex, USA), CD74 (1:500, ab108393, abcam, UK, CD44 (1:400, Ab243894, abcam, UK), CXCR2 (1:100, 20634-1-AP, Wuhan Sanying, China), CXCR4 (1:500, ab181020, abcam, U.K), CXCR7 (1:800, GTX100027, GeneTex, USA) overnight at 4°C, rinsed three times with PBS, then incubated with a secondary antibody goat anti-mouse IgG-H & L (Alexa Fluor® 488) (1:800, ab150113, Abcam, U.K) or goat anti-rabbit IgG-H & L (Alexa Fluor® 594) (1:800, ab150080, Abcam, UK) at room temperature for 2 h, rinsed three times with PBS. DAPI staining solution was added to cover the cells and incubated for 5 min. After sealing and drying, the cells were placed on the Nikon fluorescence microscope for observation.

H1 and H9 cells were cultured normally and total protein was extracted with RIPA high-efficiency lysate. The protein concentration was measured according to the instructions of BCA Protein Quantification Kit (G2026-200T, Servicebio, China). A pipetting gun was used to accurately add proteins of the same mass into the compression gel and the voltage and current conditions were determined according to the molecular weight of different proteins. The protein was separated at 120 V voltage for some time and transferred to the PVDF membrane. The membrane was kept at 250mA constant for 90 min, sealed with sealing solution (P0023B-100mL, Beyotime, China), and incubated with antibody MIF (1:1000, sc-53915, Santa Cruz, USA), CD74 (1:1000, ab108393, Abcam, UK), CD44 (1:1000, Ab243894, Abcam, UK), CXCR2 (1:1000, 20634-1-AP, Proteintech, China), CXCR4 (1:1000, Ab181020, Abcam, UK), CXCR7 (1:800, GTX100027, GeneTex, USA) at 4°C overnight, rinsed with PBS for three times, and then incubated with horseradish peroxidase (HRP) -conjugated secondary antibody goat anti-rabbit IgG (H+L) (1:1000, A0208, Beyotime, China) or goat anti-mouse IgG (H+L) (1:1000, A0216, Beyotime, China) at room temperature for 2 h. A hypersensitive ECL chemiluminescence kit (P0018S, Beyotime, China) was used to detect protein bands, and the relative protein content of each group was analyzed by ImageJ software.