Validating α-Synuclein Pathology in Organotypic Hippocampal Slices

Organotypic hippocampal culture slices (OHCS) were created from C57BL/6J pups on post-natal day 7 according to Stoppini et al. [51 (link)]. In order to validate the ability of the mouse full-length (1-140) α-Syn PFF for seeding aggregation before using them for in vivo experiments, the PFF were injected into the dentate gyrus of the OHCS, as previously described [18 (link)]. Slices were fixed 7 days post injection (dpi), and stained for pathological aggregates using conformation-specific α-Syn antibody MJF-14 (rabbit mAb MJF-14-6-4-2, 1:25,000, Abcam #ab209538) and pSer129 (mouse mAb 11A5, 1:10,000, kindly provided by ImagoPharmaceuticals), as described previously [18 (link)]. Alexa Fluor 488 anti-rabbit and Alexa Fluor 568 anti-mouse (Invitrogen, #A11008 and #A11004, 1:2000) were used for detection, along with 4′,6-diamidino-2-phenylindole (DAPI, TH.GEYER, 5 µg/mL) for staining nuclei. As negative controls, C57BL/6J OHCS were injected with either sterile PBS or monomeric α-Syn and processed as above. Furthermore, α-Syn knockout OHCS were injected with human S129A PFF and processed as above.