Briefly, cultured neurons at day in vitro 10 were fixed with 4% (w/v) paraformaldehyde for 30 minutes at room temperature and the following primary antibodies (all from Synaptic Systems) were used: rabbit anti-GFP (1∶1000), guinea pig anti-vesicular glutamate transporter-1 (1∶1000), rabbit anti-synaptophyin-1 (1∶1000), and mouse anti-synaptotagmin-1 (clone 41.1, 1∶200).
For the identification of ESC markers in undifferentiated AFiPSCs (lines 4, 5, 6, 10 and 41) and detection of lineage markers in AFiPSCs differentiated in vitro, cells were fixed, permeabilized and stained for immunofluorescent imaging as described by Prigione et al. [31].
After 7 days in vitro, cells were fixed with 4% PFA for 20 minutes at room temperature and then washed with PBS.
For RGCs in vitro, cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes, permeabilized with 0.2% Triton X-100 in PBS (T-PBS), before the TUNEL reaction.
Hepatic tissues harvested from animal experiments or cells from the in vitro studies were fixed in RNAlate solution (Ambion, Inc., Austin, TX, USA).
For the analysis of axonal length, neurons were transfected with GFP-plasmids after 2 days in vitro and were fixed in 4% paraformaldehyde after a further 24-hour incubation.
Cells from the in vitro cultures were fixed with 3.7% formaldehyde in Ca2+- and Mg2+-free PBS for 10 min and subsequently for 1 min with a 50/50 (v/v) solution of ethanol/acetone.
For in vitro study, cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature.
Five hours after the in vitro placement, brains were fixed by immersion in a 4% paraformaldehyde solution in phosphate buffer (PB; 0.1 M, pH 7.4) for one day.
In vitro neuronal cultures were fixed in 4% PFA before staining.
