Ab218810

Treated and untreated red blood cells suspension (200 μL, about 2 × 10⁷ total RBCs) were incubated for 60 min at room temperature with 100 μL diluted anti-CD36 monoclonal antibody (EPR6573, ab133625), anti-CD71 monoclonal antibody (EPR20584, ab214039), or anti-CD47 monoclonal antibody (EPR21794, ab218810) at a concentration of 5 μg/mL (all antibodies from ABCAM, Cambridge, MA, USA). These preparations then were washed twice in PBS and incubated with secondary fluorescent antibody. In addition, another sample was incubated with Annexin V-FITC Apoptosis Staining at 10 μg/mL (ab273273, ABCAM). Positive quantification was completed by hemocytometer.

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.

The specimens were fixed in 10% neutral buffered formalin and subsequently embedded with paraffin. Histological sections of 5 mm were taken from each case of ovarian tissue. Each tissue had five serial sections. Sections were deparaffinized with xylene and rehydrated for further hematoxylin–eosin staining and IHC. The expression of CD47, PD-L1, and Ki-67 in EOC tissues was detected by IHC. Breast cancer, gastric cancer, and colon cancer tissues were used as positive controls for CD47, PD-L1, and Ki-67, respectively. Negative controls were incubated with phosphate buffers instead of primary antibodies. Four primary antibodies are anti-CD47 (Cat: ab218810, diluted 1:1000, Abcam), anti-PD-L1 (Cat: ab213524, diluted 1:100, Abcam), and anti-Ki-67 (Cat: ab16667, diluted 1:200, Abcam). The experimental procedure was performed in strict accordance with the manufacturer’s instructions.

The DGs tissues were fixed, dehydrated, and paraffin-embedded. The 4 μm sections were deparaffinized and rehydrated in 100, 95, and 75% ethanol. The antigen retrieval solution with EDTA (pH 9.0) was used for PD-L1 staining, while the solution with sodium citrate (pH 6.0) for B7-H3 and CD47 staining. After endogenous peroxidase activity blocking, the sections were rinsed and incubated with the primary antibodies including anti-human PD-L1 (1:1,000, Abcam, ab228462, Cambridge, MA, USA), CD47 (1:2000, Abcam, ab218810) and B7-H3 (1:2,000, Abcam, ab219648) overnight at 4°C. TILs were stained by the CD45 (1:1,000, Abcam, ab40763) antibody, and TAMs were stained by the CD68 antibody (1:1,000, Abcam, ab213363). Images were acquired after the incubation with secondary antibodies and DAB, and counterstained with Hematoxylin. The expression level of three immune checkpoint molecules was determined by the percentage of positive cells and the staining intensity: low (negative intensity and intensity 1, and intensity 2 with positive cells < 10%) and high (intensity 2 with positive cells ≥ 10% and intensity 3) expression (19 (link)). The expression of TILs/TAMs was similarly evaluated as previously described (20 (link), 21 (link)).