Mouse MCAM/CD146 Antibody

Detection of Human and Mouse MCAM/CD146 by Western Blot. Western blot shows lysates of Bowes human melanoma cell line and mouse embryo tissue. PVDF Membrane was probed with 1 µg/mL of Sheep Anti-Mouse MCAM/CD146 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6106) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for MCAM/CD146 at approximately 117 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.

MCAM/CD146 in B16‑F1 Mouse Cell Line. MCAM/CD146 was detected in immersion fixed B16-F1 mouse melanoma cell line using Sheep Anti-Mouse MCAM/CD146 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6106) at 1.7 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Specific staining was localized to plasma membranes. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Detection of Mouse MCAM/CD146 by Simple Western^TM. Simple Western lane view shows lysates of mouse embryo tissue (15 d.p.c.), loaded at 0.2 mg/mL. A specific band was detected for MCAM/CD146 at approximately 138 kDa (as indicated) using 10 µg/mL of Sheep Anti-Mouse MCAM/CD146 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6106) followed by 1:50 dilution of HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Mouse MCAM/CD146 by Immunocytochemistry/Immunofluorescence Perivascular cells and subaortic mesenchyme are the main source of BMPER within the AGM region. (A) Distribution of BMPER protein in a transverse section of E10.5 AGM measured by immunostaining. Green, CD31; magenta, BMPER; cyan, RUNX1; blue, DAPI. gut, hindgut; nc, notochord. Bar, 50 µm. (B) Bmper mRNA in transverse section of the E10.5 AGM region by in situ hybridization. Bar, 50 µm. (C) Quantification of the mean immunostaining signal intensity (mean gray values) of DAPI (blue) and BMPER (magenta) along a box (not depicted) drawn over the dorsal-ventral axis of the AGM region from A. Distance is from the notochord (dorsal), position 0, to the intersection with the gut and the AGM region (ventral), position 500. (D) Higher magnification of the highlighted region from A showing the aortic endothelium and perivascular population. Green, CD31; magenta, BMPER; cyan, RUNX1; blue, DAPI. Bar, 50 µm. (E) Higher magnification of the region highlighted in B showing Bmper mRNA around the lining of the aorta. Bar, 50 µm. (F) Expression level of Bmper relative to Tbp in each sorted population: Lin−VC−CD45+, representing hematopoietic cells; Lin−VC+CD45−, endothelial cells; Lin−VC−CD45−CD146+, putative perivascular cells; and Lin−VC−CD45−CD146−, remaining stroma. Expression was normalized to the Lin−VC−CD45−CD146+ population. Each population as percentage of Lin− cells indicated below. Sorting was performed twice, first from one pool of embryos from four to five litters and second from two pools of embryos from four to five litters. Error bars represent SD from the mean (n = 3). Significance calculated by t test: **, P = 0.0016; ***, perivascular versus stroma, P = 0.0006; perivascular versus hematopoietic, P = 0.0002. (G) The distribution of nonendothelial, CD146-positive cells and endothelial CD146-positive cells in transverse section of the E10.5 AGM region. Green, CD31; red, CD146; blue, DAPI. Bar, 50 µm. (H) Higher magnification view of the region highlighted in G showing CD31- and CD146-positive endothelial layer and the CD146-positive CD31-negative perivascular layer around the dorsal aorta. Green, CD31; red, CD146; blue, DAPI. Bar, 50 µm. (I) Expression level of Bmper relative to Tbp in each sorted population: Lin−VC+CD45−CD43−CD41lo proHSC, Lin−VC+CD43+CD41+ type I preHSC, and Lin−VC+CD45+ type II preHSC and Lin−VC−CD45−CD146+ putative perivascular cells. Each population as percentage of Lin− cells indicated below. Sorting was performed twice, from pools of two and six litters, respectively. Expression was normalized to the Lin−VC−CD45−CD146+ population. Error bars represent SD from the mean (n = 2). Significance calculated by t test: *, perivascular versus preHSCI, P = 0.04; perivascular versus preHSCII, P = 0.03. (J) Higher magnification view of highlighted region from A showing the localization of BMPER protein in the hematopoietic clusters of the E10.5 dorsal aorta in sections measured by immunostaining. Magenta, BMPER; green, CD31; cyan, RUNX1; blue, DAPI. Bar, 50 µm. (K and L) Higher magnification view of highlighted region from B (K) and from Fig. S3 C (L) showing Bmper mRNA in some but not all cells of the intra-aortic cluster by in situ hybridization. Bars, 50 µm. Image collected and cropped by CiteAb from the following publication (https://rupress.org/jem/article/214/12/3731/42286/A-molecular-roadmap-of-the-AGM-region-reveals), licensed under a CC-BY license. Not internally tested by R&D Systems.