Goat antirabbit alexaflour 488

After 24 h, the scaffolds were washed over 15 min in a warmed 4% paraformaldehyde fixative, then rinsed (three times, 5 min for each rinse) in ice-chilled PBS. Cells cultured on scaffolds were blocked and permeabilized for 15 min with a 0.01% (v/v) Triton-X 100 (Sigma), 5% (wt/vol) BSA, and saline buffered with tris and maintained at a pH of 7.4 (Tris-buffered saline (TBS), ThermoFisher), then replaced with a solution containing primary antibodies (for at least 12 h at 4 °C). The solution containing primary antibodies was comprised of 0.1% (v/v) Tween-20 (P1379, Sigma-Aldrich), 5% (wt/vol) BSA, and Tris-buffered saline maintained at a pH of 7.4 with anti-GFAP antibody (1:800 dilution) and RT-97-S antibody (1:400 dilution) for neurofilament (64 μg/mL, Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa, Iowa City, IA). Scaffolds were washed 3 × 5 min in a solution consisting of TBS and 0.1% (v/v) Tween-20, before incubation in the secondary antibody solution: donkey antimouse AlexaFlour 594 (2 mg/mL, Life Technologies) antibody, goat antirabbit (AlexaFlour 488, Life Technologies) antibody, and DAPI (1 mg/mL, ThermoFisher) were all placed in a solution consisting of TBS, 0.1% (v/v) Tween20, and 5% (wt/vol) BSA at a 1:1000 dilution. The secondary antibody was incubated at room temperature for 1 h, then washed (three times, 5 min each wash) in PBS, before imaging.