Human ICAM-1/CD54 Antibody

ICAM‑1/CD54 in C32 Human Cell Line. ICAM‑1/CD54 was detected in live unfixed C32 human melanoma cell line using Mouse Anti-Human ICAM‑1/CD54 Monoclonal Antibody (Catalog # BBA3) at 5 µg/mL for 1 hour at 2° - 8° C. Cells were then fixed with 4% formaldehyde followed by incubation with the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; NL007) and counterstained with DAPI (blue). Specific staining was localized to cell surface and cytoplasm.

Detection of Human ICAM‑1/CD54 by Western Blot. Western blot shows lysates of C32. PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human ICAM‑1/CD54 Monoclonal Antibody (Catalog # BBA3) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for ICAM‑1/CD54 at approximately 85 kDa (as indicated). This experiment was conducted under non-reducing conditions and using Western Blot Buffer Group 1.

Detection of ICAM‑1/CD54 in Human PBMCs by Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) were stained with (A) Mouse Anti-Human ICAM-1/CD54 Monoclonal Antibody (Catalog # BBA3) or (B) Mouse IgG1 Isotype Control (MAB002) followed by anti-Mouse IgG PE-conjugated Secondary Antibody (F0102B) and Mouse Anti-Human CD14 APC-conjugated Monoclonal Antibody (FAB3832A). Staining was performed using our Staining Membrane-associated Proteins protocol.

Detection of ICAM-1/CD54 by Immunohistochemistry SERS-BFNP molecular imaging of atherosclerotic coronary arteries. A single human coronary artery was isolated from the heart of a patient undergoing heart transplantation surgery. The lumen of the artery segment was then injected with a mixture of anti-ICAM-1, anti-VCAM-1, anti-P-selectin, and isotype control BFNP, sutured closed, and incubated at 37 °C/5% CO2 for 12 h. Sutures were then removed and the artery segment was thoroughly washed prior to SERS spectroscopy and subsequent analysis of morphology, expression of adhesion molecules and SERS mapping. (B) Immunofluorescence staining for CD31, and expression of (C) ICAM-1, VCAM-1 and P-selectin are shown in red. Nuclei were counterstained using Hoechst 33342 (blue). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30613292), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ICAM-1/CD54 by Immunohistochemistry In vivo SERS-BFNP molecular imaging of adhesion molecules. Following engraftment of human adipose, HANSG mice were allowed to recover for 3 weeks. Mice were then injected intravenously with 5 μg of human recombinant TNF-alpha 4 h prior to receiving an intravenous injection of BFNP. (A) Following SERS-BFNP molecular imaging, adipose grafts were excised and immunofluorescently stained for human (red) and murine (green) CD31. (B) Isotype control, ICAM-1, VCAM-1, and P-selectin staining are also shown in white counterstained with human CD31 (red). Nuclei were counterstained using Hoechst 33342 (blue). (C) To conduct SERS-BFNP molecular imaging, HANSG mice were anaesthetized and their adipose grafts non-invasively analyzed in vivo using SERS spectroscopy. (D) SERS spectra were acquired from mice that received a mixture of isotype-PPY, -BPE and -PYOT (blue spectra), or anti-P-selectin-PPY, anti-ICAM-1-BPE, anti-VCAM-1-PYOT BFNP (red spectra). The spectra shown are from 5 isotype vs. 5 targeted mice, with each spectrum acquired from a different mouse. (E) In addition to immunofluorescence microscopy, excised adipose grafts were analyzed using SERS microscopy. Detection of BFNP from sections of adipose tissue isolated from HANSG mice that received anti-ICAM-1 (purple), anti-VCAM-1 (red), and anti-P-selectin (blue) (upper panels) or Isotype-BPE (purple), Isotype-PYOT (red), and Isotype-PPY (blue) (lower panels) are shown superimposed on darkfield tissue images alongside a magnified image of Raman maps from the scanned areas (black boxes). The colored circles in the Raman map ((E) upper panel) correlate to the acquired spectra shown in (F) above their respective reference spectra. The optical image in (E) is a darkfield image. Scale bars: (A) = 1000 μm; (B) = 100 μm; (E) = 20 μm. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30613292), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ICAM-1/CD54 by Immunocytochemistry/ Immunofluorescence Following stimulation, coronary artery endothelial cells (CAEC) express adhesion molecules detectable via immuno-SERS imaging in single and multiplex formats. (A) Fluorescence images of immunohistochemical staining of ICAM-1, VCAM-1 and P-selectin on CAEC in unstimulated and 10 ng/mL TNF-alpha -stimulated conditions. Isotype control, ICAM-1, VCAM-1 and P-selectin staining shown in green; nuclei were counterstained using Hoechst 33342 (blue). (B) CAEC were stimulated with 10 ng/mL TNF-alpha for 24 h, fixed in acetone, and incubated with isotype control, anti-ICAM-1, anti-VCAM-1 or anti-P-selectin BFNP or (C) with all BFNP simultaneously before being subjected to SERS mapping. (D) Representative spectra from anti-ICAM-1 (purple), anti-VCAM-1 (red) and anti-P-selectin (blue) BFNP acquired from the color-matched circles in (C) are shown above their respective reference spectra. Optical images in (B-C) are darkfield images. Scale bars = 20 μm. Results are representative of 3 independent experiments. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30613292), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ICAM-1/CD54 by Western Blot Methotrexate (MTX) and Ad repress TNF-alpha -induced pro-inflammatory genes without affecting primary MIR181A-1 and MIR181B-1 expression.(A) Western blot analysis of VCAM-1, ICAM-1, and E-Selectin in HUVECs treated with or without MTX (10 µM) or Ad (50 µM), after stimulation of TNF-alpha (10 ng/ml) for 8 hr. Quantification of n = 3 independent experiments. (B) Real-time qPCR analysis of VCAM-1, ICAM-1, and E-Selectin in HUVECs treated with or without MTX (10 µM) or Ad (50 µM), after treatment with TNF-alpha (10 ng/ml) for 4 hr. Real-time qPCR analysis of (C) primary transcript of MIR181A1 or (D) primary transcript of MIR181B-1 in HUVECs treated with or without MTX (10 µM) or Ad (50 µM), after treatment with TNF-alpha (10 ng/ml) for 4 hr. (A–D), n = 3–6. *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001. All values represent mean ± SEM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33416495), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ICAM-1/CD54 by Western Blot Induction of MIR181B expression by methotrexate (MTX) or adenosine is adenosine receptor A3 (ADORA3) dependent.(A) Knockdown for adenosine receptors A1, A2A, A2B, and A3 in HUVECs was performed to analyze MIR181B expression. three biological replicates. One-way ANOVA. (B) MIR181B expression in HUVECs transfected with Ctl-siRNA or ADORA3 siRNA after treatment with MTX (10 µM) or Ad (50 µM) or (C) treatment with TNF-alpha (10 ng/ml) alone or in combination MTX (10 µM) or Ad (50 µM). Three biological replicates. One-way ANOVA and Unpaired two-tailed Student t test. (D) Western blot analyses of VCAM-1, ICAM-1, and E-Selectin expression in HUVECs transfected with Ctl-siRNA or ADORA3 siRNA in the presence of TNF-alpha (10 ng/ml) in combination with either MTX (10 µM) or Ad (50 µM). Three biological replicates. Unpaired two-tailed Student t test. (E) in the presence of miRNA negative control (NS-m) or MIR181B mimics (181b-m) stimulated with TNF-alpha (10 ng/ml) or in combination with MTX (10 µM) or Ad (50 µM). Please see Figure 2—source data 1. Three biological replicates. Unpaired two-tailed Student t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. n.s. indicated non significance. All values represent mean ± SEM.Figure 2—source data 1.Knockdown efficiency for adenosine receptor siRNAs.mRNA, and protein expression analysis for (A, B) ADORA3 siRNA, (C, D) ADORA1 siRNA, (E, F) ADORA2A siRNA and (G, H) ADORA2B siRNA compared to control siRNA in HUVECs transfected for 36 hr. (A–H), n = 3. ***p<0.001. All values represent mean ± SEM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33416495), licensed under a CC-BY license. Not internally tested by R&D Systems.