Mouse anti neun clone a60

Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, washed and permeabilized in 0.1% Triton X-100 for 20 min followed by blocking in 0.05% Triton X-100 PBS buffer supplemented with 5% normal goat serum for 1 h. Cells were incubated overnight with primary antibodies as described above in addition to mouse anti-NeuN (clone A60, Millipore) and chicken anti-MAP2 (Abcam). Secondary antibodies included goat anti-mouse IgG, anti-rabbit IgG and/or anti-Chicken IgY conjugated with AlexaFluor fluorescent dyes (Invitrogen). Cells were imaged on a Perkin Elmer Opera Phenix high-content confocal imager at 20× magnification, with images processed electronically with Harmony High-Content Imaging and Analysis (PerkinElmer) and Columbus image analysis platform (PerkinElmer).

Slides were acclimated to RT and rinsed in distilled water. Heat-induced epitope retrieval was performed at 120°C using pH 6.0 buffer (Epredia, TA-135-HBL). The tissues were then blocked in 10% NGS for 1 hr. Primary antibodies were applied overnight at 4°C: Mouse Anti-NeuN (clone A60) conjugated to Alexa Fluor 555 (Millipore, MAB377A5), and Rabbit Anti-TDP-43 (Cell Signaling, 89789) or Rabbit anti-Iba1 (Wako, 019-19741) and mouse anti-GFAP conjugated to Alexa Fluor 488 (Cell Signaling, 3655) (Supplementary file 4). After rinsing, secondary antibodies were applied for 2 hr at RT: Alexa Fluor 488 Goat Anti-Rabbit IgG (Invitrogen, A32731) or Alexa Fluor 680 Goat anti-Rabbit IgG (Invitrogen 32734). All antibodies were diluted using Da Vinci Green Diluent (Biocare Medical, PD900L). Finally, slides were coverslipped using Fluorogel II with DAPI (Electron Microscopy Sciences, 17985-50).