Immunocytochemistry
Samples were fixed using a solution of 4% PFA in PBS for 20 minutes at RT. After washing with PBS (Lonza) for 5 minutes, twice, permeabilization was done through incubation of samples with a solution of 0.1% TritonTM X-100 (Sigma) in PBS for 20 minutes at room temperature. After washing samples twice with PBS for 5 minutes, samples were incubated with a blocking solution of 5% Goat Serum (DAKO) in PBS for 1 hour at room temperature. Primary antibodies against p75NTR (Millipore), fibronectin (Sigma), nestin (Millipore), GFAP (Dako), S100β (Dako) and β-III tubulin (Sigma) (Table 2) were used with a dilution of 1:200 in blocking solution and incubated for 90 minutes at room temperature. After washing with PBS, secondary antibodies (Goat anti-rabbit IgG (H+L)-Alexa Fluor 594; Goat anti-mouse (H+L)-Alexa Fluor 488, Invitrogen) and Hoechst 33342 (Molecular Probes) were diluted in blocking solution using a dilution of 1:200 and 1:1000, respectively. Images of planar culture were acquired using the microscope system EVOS® FL (ThermoFisher Scientific), while microcarrier culture 16-bit multi-color montage images were obtained with a Zeiss LSM 880 with Airscan confocal microscope system (Carl Zeiss) and Zen 2009 acquisition software (Carl Zeiss).