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URL: https://dx.doi.org/DOI:10.17504/protocols.io.syfeftn

Authors: Miriam Heinen

Summary: This staining was performed to detect fetuin-A in activated microglia. CD68 is a marker, that stains activated microglia, macrophages and monocytes. Fetuin-A and microglia were detected in paraffin sections (1 μm thickness) of formalin-fixed human brain tissue. CD68 was stained using a monoclonal mouse-anti-human antibody (Dako Cat# M0814, RRID:AB_2314148, clone KP1, dilution 1:50) and a polyclonal goat-anti-mouse Alexa Fluor® 488 conjugated secondary antibody (Thermo Fisher Scientific Cat# A-11029, RRID:AB_2534088, dilution 1:300). Fetuin-A was detected by using a monoclonal IgG2a mouse-anti-human antibody (clone MAHS-1, dilution 1.0 µg/mL), raised against purified human fetuin-A in our laboratories. Antibody binding was detected by tyramide signal amplification using a secondary biotinylated polyclonal goat-anti-mouse antibody (Dako Cat# E0433, RRID:AB_2687905, dilution 1:300) and a Tyramide Signal Amplification Kit (Life Technologies, Carlsbad, USA, T-20933). To minimize lipofuscin autofluorescence, sections were counterstained with Sudan Black (Sigma-Aldrich, Munich, Germany, 199664, dilution 0.3% in 70% ethanol, 5 minutes). Nuclei were stained with DAPI (Sigma-Aldrich, Munich, Germany D9542, dilution 0.25 µg/ml, 5 minutes). Sections were mounted with Immumount (Thermo Scientific, Waltham, USA, 9990402) and stored at 8°C in the dark. 

Associated Publications: Heinen MC, Babler A, Weis J, Elsas J, Nolte K, Kipp M, Jahnen-Dechent W, Häusler M (2018) Fetuin-A protein distribution in mature inflamed and ischemic brain tissue. PLoS ONE 13(11): e0206597. doi: 10.1371/journal.pone.0206597

Affiliations: RWTH Aachen University

External URL: https://doi.org/10.1371/journal.pone.0206597

Version: 1

Publication Date: 2018

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