Hpa012323

Briefly, cells were lysed in cell lysis buffer: 50 mM Tris-HCl (pH 8), 2% SDS, 50 mM NaCl, 1 mM EDTA, and 10% glycerol, supplemented with protease and phosphatase inhibitors (Roche), to generate total cell extracts. For the western blotting the following antibody was used: rabbit anti-CSF-1R (C-20) (Santa Cruz Biotechnology); anti-human CSF-1R antibody (1:800, HPA012323) (SIGMA-Aldrich, Milan, Italy); anti-phospho-CSF-1R(tyr723) antibody (Cell Signaling); mouse anti-TUBULIN (Santa Cruz Biotechnology) was used as a loading control. For immunoprecipitation studies, a mouse monoclonal antibody against CSF-1R was used (D-8, Santa Cruz Biotechnology). For the chemiluminescent detection of the secondary antibodies, a Western Bright ECL HRP substrate (ADVANSTA, Menlo Park, CA, USA) was used.

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