Anti goat minus

Cells were prepared as described in the immunofluorescence section and incubated with anti-EZH2 (Goat) and anti-PCNA (mouse) primary antibodies following the manufacturer’s instructions (Sigma-Aldrich). Briefly, cells were incubated for 60 min at 37 °C with anti-mouse PLUS (Sigma-Aldrich, DUO92001) and anti-Goat MINUS (Sigma-Aldrich, DUO92006), washed twice with Buffer A (Sigma-Aldrich, DUO82047), and then incubated with the ligation solution (Sigma-Aldrich, DUO92014) for 30 min at 37 °C. After ligation, cells were washed twice with Buffer A and then incubated for 100 min at 37 °C with amplification reagents (Sigma-Aldrich, DUO92014). Finally, the cells were washed three times with Buffer B (Sigma-Aldrich, DUO82048), stained, and mounted with mounting medium (Sigma-Aldrich, DUO82040) to visualize the nuclei. Images were captured with a Nikon Eclipse 300 fluorescence microscope (CompixInc).

Protein interactions between GHSR/DRD1 in mouse brain slices were detected using Duolink PLA detection kits (Sigma-Aldrich, DUO92008) following manufacturer’s instructions. The following primary antibodies were used: GHS-R1a (Santa Cruz Biotechnology, sc-10359, 1:100) and anti-DRD1 (Abcam, ab81296, 1:200). The specificity of antibodies to GHSR and DRD1 was validated as previously described (17 (link)). The following Duolink in Situ PLA Probes were used: anti–rabbit PLUS (Sigma-Aldrich, DUO92002) and anti–goat MINUS (Sigma-Aldrich, DUO92006). Images were collected on a Nikon Ti2 confocal microscope. The mean intensity of PLA⁺ signals was analyzed using Nikon-Elements Advanced Research software.

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.