Immunofluorescence staining protocol for co-staining of fetuin-A and CD68 in older human autopsy tissue v1

Abstract

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.