Sc 12767

For the detection of fluorescent proteins after infection, free-floating sections were washed three times in phosphate-buffered saline (PBS), blocked in PBS containing 4.5% bovine saline albumin (BSA) for 1 h, then incubated overnight at 4°C in PBS containing 3% BSA, 0.2% Triton X-100, and one of the following antibodies of interest. Sections were rinsed three times in PBS and incubated with Alexa Fluor 488-labeled antimouse or rabbit IgG or Alexa Fluor 555-labeled antimouse or rabbit IgG (Invitrogen) at room temperature for 1 h. The sections were rinsed in PBS and the nuclei were counterstained with DAPI (dilution 1:10,000, Wako) for 3 min. The sections were mounted in Mowiol.
Series of sections were stained for aSyn to localize the injection site in aSyn-injected rats. Tyrosine hydroxylase (TH) (Rabbit, Millipore, ab152) immunostaining was used in order to quantify the loss of dopaminergic neurons in the injected rats. The following antibodies were used to detect specific synucleins: N-terminal rat aSyn (Mouse, Bd Transduction Laboratories, 610787), recognized amino acid 121–125 of human aSyn (Mouse, Santa-Cruz, SC-12767), pS129 aSyn (Rabbit, Abcam, ab51253), and aggregated forms of aSyn (Mouse, Millipore, clone 5G4, MABN389).

Microglia were plated on 10-cm dish with F12/DMEM containing 10% FBS at a 70–80% confluent overnight, then media were changed to F12/DMEM free of FBS and stimulated with 250 nM α-Syn for 15 min, 30 min, 1 h, and 2 h. Cell protein was extracted by 1% NP-40 (Beyotime, ST366) in PBS containing 1% PIC (Pierce, 87786). Cells were lysed and collected into a 1.5 ml tube and centrifuged at 14,000 rpm at 4 °C for 5 min. The supernatants of whole cell lysates were regarded as pre-IP or input. Further, 10 μg mouse anti-human α-Syn antibody (Santa Cruz sc-12767) or mouse control IgG (Abcam, ab18447) was incubated overnight with 1000 μg protein at 4 °C overnight. Then, 100 μl Protein A + G Agarose (Beyotime, P2012) was washed twice with 200 μl 1% NP-40 in PBS, centrifuged at 14,000 rpm, 4 °C for 2min. The beads were added to protein and antibody reaction system and rocked at 4 °C for 6 h, collected by centrifugation at 14,000 rpm, 4 °C for 2 min, and then washed twice with 1% NP-40 in PBS. The protein-antibody complex was then loaded onto SDS-PAGE. Rabbit ERK (1:1000, Abcam, ab184699) and rabbit anti-human α-Syn (1:1000, Abcam, ab138501) were used to detect the specific protein band on PVDF membrane.