Immunohistochemistry of DLB Midbrain

Immunohistochemistry of midbrain sections of a patient with DLB was performed as described previously [62 (link)]. In brief, 5 μm thick paraffin-embed human midbrain sections were de-paraffinized by xylol and a descending series of alcohols and subjected to antigen retrieval by steaming in dH₂O for 30 min. Sections were blocked with 5 % goat serum in PBS-T for 1 h and incubated with primary antibodies mouse-anti- α-synuclein( 1:100, 4B12, Covance, Princeton, USA) and rabbit-anti-SOD1 (1:70, [60 (link)]) in 2,5 % goat serum at 4 °C overnight. After washing with PBS, sections were incubated with secondary antibodies (1:500, goat-anti-rabbit-Alexa568 and goat-anti-mouse-Alexa-488, both Life technology, Carlsbad, USA) in 1 % goat-serum for 1 h at RT. Subsequently sections were washed with PBS, blocked with 1 % Sudan Black (Sigma, St.Louis, USA) for 2 min and coverslipped with Fluoromont®G (SouthernBioTech, Birmingham, USA). As control, sections were stained without primary antibodies.