Anti cd164 sc 271179

Paraffin-embedded EWS specimens were incorporated in tissue microarrays (TMAs) and processed for immunohistochemistry (IHC) using an avidin–biotin–peroxidase method (Vector Laboratories, Inc., Burlingame, CA, USA). An overnight incubation with the following primary antibodies was performed: anti-CD164 (sc-271179, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:50, anti-CXCR4 (ab2074, Abcam, Cambridge, UK) diluted 1:50, anti-IGF2BP3 (sc-47893, Santa Cruz Biotechnology) diluted 1:50. Samples were classified as follows: negative, when no staining was observed; positive when weak, moderate, or strong staining was observed.

Cells seeded on fibronectin-coated coverslips (Sigma) were serum starved for 24 h and pretreated with 80 μM dynasore (S8047, Selleckchem, Houston, TX, USA), or DMSO as control, in 1% FBS medium for 30 min at 37°C. Cells were then stimulated with CXCL12 (100 ng/ml, Abcam) in 1% FBS medium for 5 min at 37°C. Cells were fixed in 4% paraformaldehyde, permeabilized in Triton X-100 0.15%-PBS, blocked in 4% BSA and incubated with the following primary antibodies: anti-CXCR4 (ab124824, dilution 1:100, Abcam); anti-CD164 (sc-271179, dilution 1:50, Santa Cruz Biotechnologies). Anti-rabbit rhodamine (#31686, dilution 1:100, Thermo Scientific) and anti-mouse FITC (#31569, dilution 1:100, Thermo Scientific) were employed as secondary antibodies. Nuclei were counterstained with Hoechst 33256 (Sigma). Confocal analysis was performed using Nikon A1R confocal microscope with a Plan Apo 60x/NA 1.4 DIC N2 objective (Nikon, Minato, Tokyo, JP). To determine colocalization of the proteins of interest, Z-stacks were acquired at 0.25 μm intervals using the following settings: 1,024 × 1,024 pixel, 2 scanner zoom, 0.5 μm scan speed. Images were analyzed using Nis Elements AR4.20.01 software (Nikon, Minato, Tokyo, JP). Colocalization was quantified by Mander's Colocalization Coefficient as we previously performed (31 (link)).