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).