Anti cd117

ST were routinely fixed and embedded in paraffin. Deparaffinized sections were cooked to perform antigen retrieval when required. Slides were subsequently stained with an automated immunostainer (TechMate 500 Plus; Dako, Cambridge, UK) using the following monoclonal antibodies: anti-CD3 (clone PS1; Novocastra, Newcastle, UK) for T-lymphocytes, anti-CD20 (clone L26; Dako) for B-lymphocytes, anti-CD68 (clone KP-1; Dako) for CD68 + macrophages, anti-CD117 (rabbit anti-human polyclonal antibody; Dako) for mast cells, anti-Hsp47 monoclonal antibodies (IgG2b M16.10A1 clone; Assay Designs) for synovial fibroblasts, anti-CD31 (clone JC70A; Dako) for endothelial cells, and anti-basic fibroblast growth factor (bFGF) (polyclonal SC-79, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-CXCL12 (clone K15C [22 (link)]) for angiogenic markers, which were significantly increased in serum from these patients in our previous study [20 (link)]. As a negative control, the primary antibodies were substituted by isotype-matched and concentration-matched control antibodies. The primary antibodies were subsequently detected by an avidin-biotin-peroxidase-based method (Envision System; Dako) and an aminoethylcarbazole color reaction (Sigma-Aldrich, St. Louis, MO, USA) as previously described [23 (link)]. Finally, the slides were counterstained with hematoxylin.

Proteins were extracted with RIPA buffer, separated on 10% SDS-PAGE, transferred to membranes (Millipore, Bedford, MA), and immunoblotted with specific antibodies. The explanted tumor was embedded in paraffin and cut into 4 mm sections using a microtome. Sections were de-paraffinized using Roticlear (Carl Roth, Germany) and hydrated in an ethanol series. Antigens were retrieved by heating the samples in 10 mM citrate buffer. The samples were blocked and then incubated at 4 °C for 16 h with the primary antibodies: anti-P63 (1:1000, BD Biosciences), anti-cytokeratin 18 (1:500, DakoCytomation), anti-cytokeratin 19 (1:500, Miltenyi Biotech), anti-CD117 (1:2000, Dako), or anti-CD44 (1:1000, Dako), and then with secondary antibody.