Nb300 141

Powder of Minimum Essential Medium (MEM), fetal bovine serum (FBS), horse serum (HS), penicillin-streptomycin (PS), B-27™ supplement, L-glutamine (L-Gln), Antibiotic-Antimycotic (AA), TRIzol reagent, Lipofectamine 2000, Alexa Fluor 488, 555 IgG secondary antibodies, and 4′, 6′-Diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, USA). Neurobasal^® medium, Eagle’s basal medium (BME), and N-2 Supplement were purchased from Gibco (Grand Island, NY, USA). Poly-l-lysine (PLL), glutamate, Cytosine-β-d-arabinofuranoside (AraC), bovine serum albumin (BSA), sucrose, formaldehyde solution, chloroform, phenol solution, and IWR-1 were from Sigma-Aldrich (Saint Louis, MO, USA). WNT3A recombinant protein was purchased from PeproTech (Rocky Hill, NJ, USA). WNT8A and WNT9B were from R&D Systems (Minneapolis, MN, USA). Power SYBR Green Master Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-Tau (sc-5587) and anti-MAP2 (sc-56561) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-TUJ1 antibodies (802001 and 801202) were purchased from BioLegend (San Diego, CA, USA). Anti-GFAP (GTX108711), anti-NeuN (GTX30773) antibodies, and rabbit IgG (GTX26702) were from GeneTex (Irvine, CA, USA) or Novus (NB300-141, Centennial, CO, Canada) for cryosection staining.

For antibody-conjugated gold staining, 10 μL of isolated exosome sample was covered by a copper grid film (Electron Microscopy Sciences, USA) for 20 min of absorption. The grids were then incubated with a 100 μL drop of 50 mM glycine PBS solution for 10 min, followed by PBS washing. 1% BSA PBS solution was then used for blocking grids. Primary antibodies—mouse anti-CD63 (NBP2-32829, Novus Bio., USA) and rabbit anti-GFAP (NB300-141, Novus Bio., USA)—were independently used to stain the grids for 30 min, followed by PBS washing. The grids were stained with 10 nm gold-conjugated goat anti-rabbit IgG (Electron Microscopy Sciences, USA) and 15 nm gold-conjugated goat anti-mouse IgG (Electron Microscopy Sciences, USA) secondary antibodies for 30 min and then washed with PBS. The grids were then incubated with uranyl–acetate solution for 10 min followed by water washing for complete negative staining. An electron microscope (FEI Company, USA) was used to image the grids.