Polymer horseradish peroxidase conjugated secondary antibody

H&E staining and IHC were performed as previously described [36 (link)]. The brain samples were embedded in paraffin blocks, and the sections were prepared by HistoCore AutoCut (Leica, Deerfield, IL, USA). Next, the sections were cut into 4 μm sections and stained with H&E, following standard procedures. For IHC, sections were treated with 3% hydrogen peroxide/methanol and then with 0.25% pepsin to retrieve antigens. Next, samples were incubated in blocking solution (Dako, Carpinteria, CA, USA), after which they were incubated at 4 °C overnight with the specific primary antibodies diluted in the antibody diluent (Dako). The sections were subsequently washed with tris buffered saline with 0.1% Tween 20 and then incubated with polymer-horseradish peroxidase-conjugated secondary antibody (Dako). A 3,3′-diaminobenzidine substrate chromogen system (Dako) was utilized to detect antibody binding. Stained sections were observed under an Olympus IX71 inverted microscope (Olympus Optical, Tokyo, Japan).