Quantifying Synuclein Neuropathology in Mouse Brains

Mouse brains were fixed in 10% (vol/vol) formalin, processed, embedded, and sectioned as previously described [25 (link)]. Brains were cut into four sections prior to processing through graded alcohols, clearing with xylene, infiltrating with paraffin, and embedding. After deparaffinization, sections were exposed to heat-mediated antigen retrieval with citrate buffer (0.1 M, pH 6) for 20 min. Slides were stained overnight at room temperature after blocking in 10% (vol/vol) normal goat serum using EP1536Y (pS129 α-synuclein; 1:1,000; Abcam), p62 (Anti-SQSTM1; 1:1,000; Abcam), and glial fibrillary acidic protein (GFAP; 1:500; Abcam) primary antibodies. Secondary antibodies conjugated to AlexaFluor 488, 568, or 647 (Thermo Fisher) were used to detect immunolabeling.
Slides were imaged using the Zeiss AxioScan.Z1. Digital images were analyzed using the Zen Analysis software package (Zeiss). To quantify α-synuclein neuropathology, a pixel intensity threshold was determined using a positive control slide and was then applied to all slides. Regions of interest were drawn around the HC, Thal, HTH, midbrain (Mid), and pons. The percentage of pixels positive for staining in each region was determined.