Anti ki67 antibody

Macrophages were obtained from BM precursor cells. In brief, total BM was obtained from mice by flushing femur and tibia BM with DMEM. Cells were cultured in a DMEM-containing conditioned medium of L929 cell line (enriched in M-CSF) for 7 days. After washing, DMEM-serum-free medium was added for 24 h, and cells were recovered and centrifuged in order to obtain macrophage-conditioned medium. For phagocytosis studies, cell cultures were performed under standard conditions in DMEM/F12 (Gibco) containing 20% FBS and 2% Ultroser G serum (Pall) during 16 h with or without addition of recombinant recombinant human cytokines CX3CL1 (R&D Systems) or CCL2 (PeproTech) at 100 nM.
Murine MPCs were obtained from TA muscle and cultured with standard conditions in DMEM/F12 (Gibco) containing 20% FBS and 2% Ultroser G serum (Pall). For proliferation studies, MPCs were seeded at 10,000 cells per cm² on Matrigel (1/10) and incubated for 1 day with macrophage-conditioned medium+2.5% FBS. Then, cells were incubated with anti-ki67 antibodies (15580, Abcam). For fusion index studies, MPCs were seeded at 30,000 cells per cm² on Matrigel (1/10) and incubated for 3 days with macrophage-conditioned medium containing 2% horse serum. Then, cells were incubated with antibodies against desmin (32362, Abcam).

Zymosan A (ZyA) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Cytokines and platelet-derived growth factor (PDGF) were from R&D Systems (Minneapolis, MN, USA). Compound 968, 5-(3-bromo-4-(dimethylamino)phenyl)-2,2-dimethyl-2,3,5,6-tetrahydrobenzo[a]phenanthridin-4(1H)-one, was obtained from Calbiochem (La Jolla, CA, USA). Anti-glutaminase and anti-Ki-67 antibodies were obtained from Abcam (Cambridge, UK).

Paraffin-embedded, formalin-fixed tissues were immunostained for SLC7A11 and Ki-67 proteins using the avidin-biotin-peroxidase method. Briefly, paraffin sections were dewaxed in xylene and dehydrated using a graded series of alcohols. The sections were then treated with protein blockers and incubated overnight at 4°C with anti-SLC7A11 (1:200; Abcam, Cambridge, MA, USA) or anti-Ki-67 antibodies (1:100; Abcam, Cambridge, MA, USA). The signal was amplified and visualized with diaminobenzidine chromogen, followed by counterstaining with hematoxylin. Finally, the sections were dehydrated using a graded series of alcohols, cleared in xylene, and mounted. Tumor cells with cytomembrane and nuclei containing brown immunoreactive products were considered to be positive for SLC7A11 and Ki-67 expression, respectively.

IHC analysis of Ki-67 levels was conducted on paraffin slides of mouse liver and metastatic lung tumor tissues using anti-Ki-67 antibodies (Abcam, Cambridge, UK) and anti-FTO (#ab126605, Abcam). The detailed description of IHC was conducted as previously reported [5 (link)].

Briefly, the subcutaneous tumor was resected and fixed in 10% formaldehyde. After incubation with anti-Ki-67 antibodies (1:200, Abcam, Cambridge, UK) , anti-SHPRH (1:100, Abcam, Cambridge, UK) and anti-E-cadherin (1:200, Abcam, Cambridge, UK) overnight, we visualize the slide using streptavidin-horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA). Then Amplification and visualization were performed using DAB (Santa Cruz Biotechnology, Santa Cruz, CA, USA) substrate chromogen solution, followed by counterstaining with hematoxylin.