Pa1 075

CML and receptor for AGEs (RAGE) immunopositivity was analyzed by immunohistochemistry on 7 μm paraffin-embedded sections of ileum and submandibular salivary glands. Slides were deparaffinized, rehydrated, and antigens were retrieved by 5 min boiling in 10 mM sodium citrate buffer, pH 6.0. After blocking, sections were incubated overnight with primary antibodies (CML, R&D, #MAB3247, dilution 1:50; RAGE, Invitrogen, #PA1-075, dilution 1:50) and subsequently for 1 h with HRP-conjugated secondary antibodies (dilution 1:200) and nuclei were counterstained with hematoxylin.

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.