Aqueous mounting media

Sections were double-labeled with a mouse anti-GFAP or anti-CD44 and a rabbit anti-CHI3L1 antibody overnight. The appropriate secondary antibody was applied [Cy3-donkey anti-rabbit IgG for CHI3L1 and Cy2-donkey anti-mouse IgM for GFAP] and incubated in a 0.1 thioflavin solution (10 min) to visualize aggregated amyloid. Auto-fluorescence was blocked with Auto-fluorescence Eliminator Reagent (Millipore) and sections cover-slipped with aqueous mounting media (Thermo Scientific). Dual immunofluorescence was visualized, and images were acquired using the Revolve Fluorescent Microscope (Echo Laboratories, San Diego, CA, USA) with excitation filters 405, 489, and 555 nm for thioflavin, Cy2, and Cy3, respectively.

Double stain was used to evaluate whether astrocytes display tau pathology using antibodies to the astrocytic marker GFAP and phosphorylated tau at S396/404, PHF-1, in the hippocampus of CTE stage IV cases [16 (link)]. One section per case was incubated overnight at room temperature with both GFAP (1:1000, DAKO Denmark) and PHF-1 (1:1000, gift from Peter Davies) after 10 min citric acid pH 6 antigen retrieval. Then, the appropriate secondary antibodies were applied for 1 h at room temperature as followed: first Cy5-conjugated donkey anti-mouse IgG for PHF-1 (1:400, Jackson Immuno-research, West Grove, PA) and secondly, after several washes, Cy2-donkey anti-rabbit IgG for GFAP (1:400, Jackson Immuno-research). DAPI, a nuclear fluorescence stain, was employed at 1:2000 at room temperature for 10 min. Auto-fluorescence was blocked with Auto-fluorescence Eliminator Reagent (Millipore, MA, USA) according to manufacturer’s instructions and cover-slipped with aqueous mounting media (Thermo Scientific). Dual immunofluorescence was visualized with the aid of a Revolve Fluorescent Microscope (Echo laboratories, CA, USA) with excitation filters for DAPI (pseudocolored green), Cy2 (emission green; pseudocolored red) and Cy5 (pseudocolored blue) as described previously [17 (link)].