Mfg e8

Manufactured by Santa Cruz Biotechnology

Sourced in Canada, United States

MFG-E8 is a protein that functions as a bridging molecule between apoptotic cells and phagocytes, facilitating the clearance of dying cells. It contains two N-terminal EGF-like domains and a C-terminal discoidin-like domain. MFG-E8 is involved in various biological processes, including immune response, cell adhesion, and tissue remodeling.

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Anti-MFG-E8 (sc-271574) (3 citations) MFG-E8 antibody (2 citations)

6 protocols using mfg e8

Immunohistochemical Analysis of Dopaminergic Neurons

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The following chemicals were purchased from Sigma‐Aldrich, St. Louis, MO: Apomorphine (A4393‐1G), 3,3′‐diaminobenzidine (DAB, D5905), ExtrAvidin Peroxidase Staining Kit (EXTRA2), Bovine serum albumine (BSA, A2058) and anti‐TH antibody (T2928), Triton X‐100 (X100), and 6‐OHDA (H116). Antibodies against Syntenin‐1, MFG‐E8, and LGR5 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti‐HSP 70 antibody was from BD Transduction Laboratories (Becton, Dickinson and Company, Franklin Lakes, NY). Artificial cerebrospinal fluid (aCSF) was prepared ex tempore. Apomorphine was dissolved in saline prior to the injection.

Narbute K., Piļipenko V., Pupure J., Dzirkale Z., Jonavičė U., Tunaitis V., Kriaučiūnaitė K., Jarmalavičiūtė A., Jansone B., Kluša V, & Pivoriūnas A. (2019). Intranasal Administration of Extracellular Vesicles Derived from Human Teeth Stem Cells Improves Motor Symptoms and Normalizes Tyrosine Hydroxylase Expression in the Substantia Nigra and Striatum of the 6‐Hydroxydopamine‐Treated Rats. Stem Cells Translational Medicine, 8(5), 490-499.

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Protein Expression Analysis by Western Blot

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Protein extraction and Western blot analysis were performed as previously described (Lauber et al., 2013 (link); Li et al., 2015 (link)). Protein samples (30ug) from the left cerebral hemisphere (perforation side) were loaded on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane. Blotting membranes were incubated for 2 hours with a blocking solution (5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20), and then incubated overnight at 4°C with the following primary antibody: MFGE8, integrin-β3 and β-Actin (Santa Cruz Biotechnology, Santa Cruz, CA), IL-1β (Abcam, Cambridge, MA), Cleaved caspase-3 (Cell Signaling Technology, Danvers, MA). Next, the membranes were incubated for 1 hour with appropriate secondary antibodies at room temperature. Lastly, the bands were visualized using the ECL Plus chemiluminescence reagent kit (Amersham Bioscience, Arlington Heights, IL) and quantified by optical density methods using the Image J software (National Institutes of Health, Bethesda, MD).

Liu F., Chen Y., Hu Q., Li B., Tang J., He Y., Guo Z., Feng H., Tang J, & Zhang J.H. (2015). MFGE8/Integrin β3 Pathway Alleviates Apoptosis and Inflammation in Early Brain Injury after Subarachnoid Hemorrhage in Rats. Experimental neurology, 272, 120-127.

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Molecular Mechanisms of MFG-E8 Regulation

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Recombinant mouse MFG-E8 was obtained from R&D systems (Minneapolis, MN, USA). Hydrogen peroxide (H₂O₂) and LY294002 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against MFG-E8, Aggrecan, TXNIP, and Caspase-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against Collagen II, MMP13, ADAMTS5, IL-1β, IL-18, NLRP3, GSDMD, PI3K, p-PI3K, AKT, p-AKT and Lamin B were purchased from Abcam (Cambridge, MA, USA). Primary antibodies against β-actin, Nrf2, SOD2, HO-1, and NQO1 were purchased from Proteintech (Rosemont, IL, USA).

Ma H., Xie C., Chen Z., He G., Dai Z., Cai H., Zhang H., Lu H., Wu H., Hu X., Zhou K., Zheng G., Xu H, & Xu C. (2022). MFG-E8 alleviates intervertebral disc degeneration by suppressing pyroptosis and extracellular matrix degradation in nucleus pulposus cells via Nrf2/TXNIP/NLRP3 axis. Cell Death Discovery, 8, 209.

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Protein Expression Analysis by Western Blot

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Equal quantities of protein extracts were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, USA). After blocking in 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: MFG-E8 (Santa Cruze), NG2 (Millipore), and PLP1 (Abclone). The membranes were subsequently incubated with corresponding secondary antibodies and visualized with chemiluminescence reagents provided with an ECL kit (Bioworld, USA).

Dong X., Zhang Z., Shu X., Zhuang Z., Liu P., Liu R., Xia S., Bao X., Xu Y, & Chen Y. (2023). MFG-E8 Alleviates Cognitive Impairments Induced by Chronic Cerebral Hypoperfusion by Phagocytosing Myelin Debris and Promoting Remyelination. Neuroscience Bulletin, 40(4), 483-499.

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Immunohistochemical Analysis of Brain Tissue

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Mice were anesthetized and transcardially perfused with cold PBS and cold 4% PFA. The dissected brains were post-fixed in 4% PFA for 24 h at 4 ℃ and subsequently cryoprotected in 30% sucrose for 72 h at 4℃. Frozen sections were cut at 20 μm for use. Cells were fixed in 4% PFA for 30 min and washed three times with PBS. Sections or cells were blocked with 5% BSA and 0.25% Triton X-100 and incubated overnight at 4 ℃ with primary antibodies. The primary antibodies were as follows: Oligo2 (Millipore), CC1 (Abcam), PDGFRα38 (BD Biosciences), MFG-E8 (Santa Cruz), Iba-1 (Abcam), GFAP (Proteintech), Map2 (Bioworlde), CD68 (Abcam), and degraded myelin basic protein (dMBP; Millipore). The next day, sections or cells were incubated with appropriate secondary antibodies at room temperature for 2 h. Fluorescence images were obtained using a fluorescence microscope (Olympus IX73) or a confocal laser-scanning microscope (Olympus FV3000).

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Protein Expression Analysis of Brain Samples after SAH

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The brain samples were collected at 24 hours after SAH. Western blotting was performed as described previously^{18 (link)}. Primary antibodies used were: MFGE8 (Santa Cruz Biotechnology), HO-1, Nrf2 (Santa Cruz Biotechnology), 4-Hydroxynonenal (4-HNE, Abcam Biotech Company), Nitrotyrosine (Santa Cruz Biotechnology), integrin β3, extracellular-signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK).

Liu F., Hu Q., Li B., Manaenko A., Chen Y., Tang J., Guo Z., Tang J, & Zhang J.H. (2014). Recombinant Milk Fat Globule–EGF Factor-8 Reduces Oxidative Stress via Integrin β3/Nuclear Factor Erythroid 2–related Factor 2/ Heme Oxygenase Pathway in Subarachnoid Hemorrhage Rats. Stroke; a journal of cerebral circulation, 45(12), 3691-3697.

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