β 3 tub

At day 14 in vitro, the cultures were fixed with 4% paraformaldehyde for 10 min, rinsed 3× with HEPES Buffered Hank’s saline (HBHS) (in g/L; 7.5 g NaCl, 0.3 g KCl, 0.06 g KH2PO4, 0.13 g Na2HPO4, 2 g Glucose, 2.4 g HEPES, 0.05 g MgCl2:6H2O, 0.05 g MgSO4:7H2O, 0.165 g CaCl2, 90 mg NaN3, at pH 7.4), then permeabilized with 0.2% Triton-X (Sigma-Aldrich, St. Louis, MO). The cultures were then blocked with 10% normal goat serum (Jackson Immunoresearch, West Grove, PA) for 1 h, after which primary antibodies to beta-3-tubulin (β-3-tub) (Covance, Princeton, NJ), which labels neurons; Glial Fibrillary Acidic Protein (GFAP) (Millipore, Billerica, MA), which labels astrocytes; and Ionized Calcium binding adaptor molecule 1 (Iba1) (Wako, Osaka, Japan), which labels microglia, were added, and the cultures incubated in a 4°C refrigerator overnight. The wells were then aspirated, rinsed in HBHS 3×, and the following secondary antibodies were added: Alexa Fluor 488 Goat anti-mouse, Alexa Fluor 555 Goat anti-chicken, and Alexa Fluor 635 Goat anti-rabbit (Invitrogen, Carlsbad, CA). After a 2 h incubation at room temperature, the secondary antibodies were rinsed 3× with HBHS, and a final volume of 100 μl of HBHS was left in the wells for imaging. Special care was taken to ensure the microwire segments remained attached to the bottom of the wells.