Ab254143

Primary cortical neurons were treated with 5 μg/mL PFF for 5 days and then incubated with 50% MCM for 24 h. The cells were blocked with 10% goat serum, and incubated with mouse anti-MAP2 (1:500, ab254143; Abcam) and rabbit anti-p-α-syn (Ser129) (1:500, 23706S; Cell Signaling Technology) at 4 °C overnight. The cells were stained with Alexa Fluor 647 goat anti-mouse (1:1,000, A32728; ThermoFisher Scientific) and Alexa Fluor 568 goat anti-rabbit (1:500, A21428; ThermoFisher Scientific), and then stained with DAPI. Fluorescent images were captured using a TCS SP8 confocal laser scanning microscope and analyzed by ImageJ software.

Immunohistochemistry of human postmortem brain tissues was completed on 10‐µm thick paraffin sections fixed with 4% paraformaldehyde. The sections were blocked with 2% bovine serum albumin in PBS for 1 h at room temperature before applying the primary antibody overnight at 4 °C. The human brain sections were stained with anti‐NeuN (ab104224, Abcam, 1:200) for neurons, anti‐GFAP (ab4648, Abcam, 1:200) for astrocytes, and anti‐PSAP (10801‐1‐AP, Proteintech, 1:200). For proliferation experiments, sections were stained with anti‐Ki67 (ab15580, Abcam, 1:200) and EdU (C10310‐3, Cell‐light EdU Apollo488, Ribobio). To study neuronal morphology, staining was performed with anti‐MAP2 (ab254143, Abcam, 1:200). To quantify the synaptic count in neurons, cells were stained with anti‐Synapsin I (ab254349, Abcam, 1:200) and anti‐PSD95 (124011, Synaptic Systems, 1:200). Sections were washed three times for 5 min each at room temperature and incubated with DAPI(C0065, Solarbio) for 10 min. The sections were then incubated with the secondary antibodies (A32723 and A11037, ThermoFisher, 1:200) for 1 h at room temperature after incubation with primary antibodies. All sections were mounted on glass slides with fluorescent mounting medium (ZLI‐9556, ZSGB‐BIO).