Epr21794

CD47 immunohistochemical staining was performed with 4 µm thick sectioned slides from TMA blocks. The Ventana BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ, USA) was used according to the manufacturer’s protocol. Anti-CD47 monoclonal antibody (1:200 dilution, EPR21794; Abcam, Cambridge, UK) was used to detect CD47 expression.

2x10⁴ MM cells were seeded on glass coverslips and the following day treated overnight with either BoxA (400 nM) or CXCL12 (10 nM) and PBS (control). Following treatment, cells were fixed with 4% paraformaldehyde in PHEM buffer for 10 min at RT, washed twice with 1% BSA in PBS for 5 min, and then blocked with 4% BSA and 10% goat serum in PBS. Cells were overlayed with the primary antibodies:
rabbit monoclonal anti‐CD47 (1:100, EPR21794, Abcam #AB218810); mouse monoclonal anti‐CD47 (1:50, B6H12, Santa Cruz #sc12730) either alone or in combination or goat polyclonal anti‐CXCR4 (1:100, Abcam #AB1670), or rabbit monoclonal anti‐TLR4 (1:50, Cell signaling #14358) and or rabbit polyclonal anti‐RAGE (1:100, Invitrogen #PA1‐075) for 1 h at room temperature. Following three washes with 0.2% BSA in PBS, the cells were incubated with secondary antibodies in 0.2% BSA/PBS + 10% goat serum and incubated for 45 min at RT. For nuclei staining, 1 µg/ml Hoechst 33358 was used. For cytosol detection, Phalloidin FITC (P5282, Sigma‐Aldrich) was used.
Secondary probes (Duolink, Sigma‐Aldrich) for PLA reaction were as follows: Anti‐Rabbit MINUS (#DUO92005), Anti‐Rabbit PLUS (#DUO92002), Anti‐Goat MINUS (#DUO92006), and Anti‐Goat PLUS (#DUO92003). When both primary antibodies were used, the PLA products were obtained by using the Anti‐Rabbit PLUS and Anti‐Goat MINUS probes.

We constructed tissue microarrays (TMA) to efficiently evaluate biomarker expression [17 (link)]. Manual microarray systems (Tissue Microarray Set, Labro, Seoul, Korea) and formalin-fixed paraffin-embedded tissues were used for tissue microarray (TMA) construction. We selected a representative area of the tumor by light microscopy and collected tissue cores (3.0 mm) from the corresponding donor block. Then, the tissue cores were transferred to a recipient block consisting of 6 × 5 samples. Each TMA block was cut into 4-μm-thick sections and stored at −70 to −80 °C.
IHC was used to evaluate CD47 expression in tissue samples of produced TMA. According to the manufacturer’s protocol, we performed IHC with the Benchmark XT automated staining system (Ventana Medical Systems, Tucson, AZ, USA), and a recombinant anti-CD47 antibody (EPR21794, Abcam, Cambridge, UK; diluted 1:200) was used as a primary antibody.