Anti mouse cd45 antibody

Tumor tissue samples embedded in Tissue-Tek optimum cutting temperature (OCT) were sectioned by a microtome-cryostat (8 μm). Formalin fixed, paraffin-embedded tissue specimen was prepared for 5 μm sections. Frozen sections were fixed in HistoChoice MB tissue Fixative solution (Ambresco, Cleveland, OH, USA). Subsequently in both frozen and paraffin-embedded sections, peroxidase activity was inhibited by 3% hydrogen peroxide. Additionally, samples were blocked using SuperBlock blocking buffer (Thermo Fisher Scientific). Anti-mouse CD45 antibody, anti-mouse Ly6G/Ly6C antibody, and anti-mouse CD3 antibody (Abcam, Cambridge, MA, USA) were used for immunohistochemistry staining. The signal was developed by diaminobenzidine (DAB) substrate (Dako, Santa Clara, CA, USA).

Freshly excised tumors were manually minced before enzymatic disaggregated for 2 hrs with Accutase (Stem Cell technologies, Vancouver, CA) and pooled. Cell proliferation was assessed by quantification with Ki-67 immunohistochemistry on cytospin sections of the disaggregated tumors with a anti-human Ki-67 antibody (1:300, clone MIB-1) (Dako, CA, USA). Positive cells were scored by visual examination of 10 randomly chosen fields containing at least 100 cells. For FACS analysis of the tumor-derived cells, anti-human CSF-1R antibody (1:200, HPA012323) (SIGMA-Aldrich, Milan, Italy) and anti-mouse CD45 antibody (1:100, ABCAM, Cambridge, UK) were used