Map2ab

The primary antibodies dilutions in this study were used at 1:1000, except when indicated. They were: MAP2 A/B (MAB5622, Millipore, Billerica, MA, United States); MAP2 A/B (MAB378, Millipore, Billerica, MA, United States); Aldo C (sc-12065, Santa Cruz Biotechnology, Dallas, TX, United States); TSG101 (Ab83, Abcam, Cambridge, MA, United States); Flotilin-1 (610821, BD, Franklin Lakes, NJ, United States); CD63 (sc-15363, Santa Cruz Biotechnology, Dallas, TX, United States); GM130 (Ab52649, Abcam, Cambridge, MA, United States); GFAP ( G2032-28B-PE, US Biological, Salem, MA, United States); GFP (Ab6673, Abcam, Cambridge, MA, United States); GFP (MAB3580, Millipore, Billerica, MA, United States) (From our lab. This antibody detects a faint band over 63 kDa in astrocyte homogenates); Alix (sc-53540, Santa Cruz Biotechnology, Dallas, TX, United States); and β-actin (A5441, Sigma-Aldrich, St Louis, MO, United States).

Neurons were differentiated on poly-D-lysine/laminin coated glass coverslips. These were fixed with 4% fresh paraformaldehyde for 20 min at room temperature before being washed three times with phosphate-buffered saline (PBS). Samples were then blocked for 1 h at room temperature with 0.3% Triton/ PBS/5% goat serum before being incubated overnight with primary antibody in 0.2% Triton/PBS/2% goat serum at 4°C. After three washes in PBS, secondary antibody (goat anti-mouse, Alexa Fluor 488 or 555, 1:1000) in PBS/Hoechst (1:4000) was next applied for 1 h at room temperature. Primary antibodies used included β-III tubulin (1:500; Sigma- Aldrich), orthodenticle homeobox 1 (OTX1), REELIN, T-Box Brain 1 [TBR1] (1:50; Developmental Studies Hybridoma Bank [DSHB], Iowa City), Forkhead Box G1 (FOXG1) (1:100; Abcam), Synapsin (1:500; Calbiochem), microtubule associated protein 2ab (MAP2ab) (1:500; Sigma-Aldrich), gamma-aminobutyric acid (GABA) (1:500; Sigma-Aldrich) and glutamate (1:500; Sigma-Aldrich).

Fixed cells on organ-chips or glass coverslips were washed in phosphate-buffered saline (PBS) and permeabilized in PBS Triton 0.2% for 10 min and blocked in PBS containing 5% donkey serum (Sigma) and Triton X-100 0.2% at 4 °C overnight. Primary antibodies were TH 1:300 (Novus Biologicals, Centennial, CO, USA, NB300-109), GFAP 1:1000 (Dako, Glostrup, Denmark, Z0334) and Map2ab 1:1000 (Sigma, M1406). Samples were washed three times in PBS and stained with species-specific Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (1:500 dilution, Invitrogen, Waltham, MA, USA) for 2 h at room temperature, followed by 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Confocal z-stack images were acquired using an A1 microscope (Nikon, Tokyo, Japan) with 10× or 20× objectives and rendered using maximum-intensity projection through FIJI.