Isomangiferin was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Its purity was verified as ≥98% by high-performance liquid chromatography. Reagents used for cell cultures such as fetal bovine serum (FBS) and trypsin, and the growth medium were all purchased from Gibco Life Technology (Carlsbad, USA). Antibodies specific for actin, VEGFR-2, phosphorylated VEGFR-2 (p-VEGFR-2), platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), protein kinase B (PKB/AKT), phosphorylated protein kinase B (p-AKT), extracellular regulated protein kinase (ERK), phosphorylated ERK (p-ERK), poly ADP-ribose polymerase (PARP), cleaved PARP, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), focal adhesion kinase (FAK), Caspase 3, and β-actin were obtained from Cell Signaling Technology (Danvers, USA). The MTS cell proliferation assay kit was purchased from BD Biosciences (Franklin Lakes, USA). VEGFR-2 signaling pathway agonist vascular endothelial growth factor (VEGF) and antagonist SU5408 were purchased from Sigma-Aldrich (St. Louis, USA).
Cd31 pecam 1
Manufactured by Cell Signaling Technology
Sourced in United States
CD31/PECAM-1 is a transmembrane glycoprotein that functions as a cell adhesion molecule. It is primarily expressed on the surface of endothelial cells and plays a role in the regulation of angiogenesis and vascular permeability.
Automatically generated - may contain errors
Lab products found in correlation
M1 like marker cd38, by Thermo Fisher Scientific (2 mentions)
M2 like marker cd 206, by Bioss Antibodies (2 mentions)
Dako reagents, by Agilent Technologies (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phosphorylated vegfr 2 p vegfr 2, by Cell Signaling Technology (1 mentions)
Anti mouse alexa488, by Abcam (1 mentions)
Anti α smooth muscle actin, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Ve cadherin, by Cell Signaling Technology (1 mentions)
Ab5694, by Abcam (1 mentions)
Sv40 large t antigen, by BD (1 mentions)
Lsm 800 microscope, by Zeiss (1 mentions)
Hoechst dye, by Thermo Fisher Scientific (1 mentions)
Nanozoomer 2.0 ht system, by Hamamatsu Photonics (1 mentions)
6 protocols using cd31 pecam 1
1
Isomangiferin Regulates Angiogenic Signaling
2
Isolation and Characterization of Uterine Vascular Smooth Muscle Cells
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UtVSMCs from pregnant rats (GD16) were isolated from the main uterine arteries using an enzymatic digestion method (24 (link)). Cell pellets were resuspended in DMEM (with 10% FBS and 100 U/mL penicillin, 100 µg/mL streptomycin), and cell solutions were distributed in culture dishes and kept in a humidified incubator at 37°C (5% CO2). Cell culture media was replaced every 48 h. Purity of utVSMCs was tested by immunocytochemistry for positive staining of VSMC marker smooth muscle actin (anti-α-smooth muscle actin, 1:400, Sigma Cat. No. A2547; secondary: anti-mouse Alexa 488, 1:500, Abcam, Cat. No. ab150113). Anti platelet endothelial cell adhesion molecule 1 (PECAM 1; CD31, 1:3,200, Cell Signaling, Cat. No. 3528S) was used as a negative control. Cultured utVSMCs were used at passage 2 to avoid genotypic and phenotypic drifts.
3
Immunohistochemical Profiling of Lung Tissue
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Fixed lung was embedded in paraffin, sectioned, and stained for immunohistochemical examination, similar to methods described (Hwang et al. 2019 (link)), diluted at 1:2000, was performed according to manufacturer’s instructions with a modification of 20 min at low pH for antigen retrival, and visualized using standard HRP techniques and DAB chromogen using Dako reagents (Dako, Agilent, Santa Clara, CA). In a similar manner, M1-like marker CD38 (Invitrogen, ThermoFisher, Cat# 14-0381-02), diluted at 1:1000, M2-like marker CD 206 (Bioss, Cat# bs-4727R), diluted at 1:1000, and endothelial cell marker CD31/PECAM-1 (Cell Signaling, Cat# 77699T) diluted at 1:200 were used for visualization on serial slide sections. Hematoxylin counterstained slides were viewed by a trained pathologist, with descriptive results obtained in an experimentally blinded manner.
4
Immunohistochemical Profiling of Lung Tissue
5
Protein Expression Analysis in MVECs
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MVECs were treated with RIPA buffer containing a cocktail of protease inhibitors (Roche, 11836153001). Proteins were extracted and quantified with a BCA protein assay kit (Thermo Scientific 23225). Samples (10 µg) were loaded onto 12% SDS-PAGE gels and transferred onto PVDF membranes. Blots were probed by anti-human vascular endothelial cadherin (VE-Cadherin) (1:1000, Cell Signaling Technology 2158S), CD31 (PECAM-1) (1:1000, Cell Signaling Technology 3528), matrix metallopeptidase-2 (MMP-2) (1:1000, Cell Signaling Technology 4022), α-SMA (1:1000, Abcam ab5694), extracellular signal-regulated kinase (ERK) (1:1000, Cell Signaling Technology 4695S), pERK (1:1000, Cell Signaling Technology 9101S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, Invitrogen MA5-15738) and visualized by chemiluminescence (ATTO), and quantified using ImageJ software.
6
Histological Analysis of Lung Metastasis
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Hematoxylin and eosin (H&E) staining was performed on paraffin-embedded 5 µm sections of lungs. Whole slide images were captured using an Aperio GT450 at the Georgetown University Histopathology and Tissue Shared Resource Core and a Hamamatsu NanoZoomer 2.0-HT System at the Washington University in St. Louis Alafi Neuroimaging Lab. Immunohistochemistry staining against SV40 Large T antigen (BD, Franklin Lakes, NJ, USA, #554150) was performed as described previously [23 (link)] on m6 lung metastases from REAR mice. Metastasis burden as a proportion of cross-sectional tissue area and metastatic foci counts was calculated using QuPath v0.3.2 [24 (link)] in a blinded manner. Immunofluorescence staining was performed as described previously [25 (link)] on paraffin-embedded 5 µm sections of lungs for CD-31/Pecam-1 (Cell Signaling Technologies, Danvers, MA, USA, #77699), GFP (Invitrogen, Waltham, MA, USA, #332600), and Hoechst dye (Fisher Scientific, Waltham, MA, USA). Slides were imaged on a Zeiss LSM800 microscope.
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