Rabbit anti pax6

NSCs were washed with PBS for three times and fixed in 4% paraformaldehyde for 15 min. The fixed cells were washed with PBS and penetrated with 0.2% Triton X-100 for 20 min. Then the cells were washed with PBS and incubated in normal goat serum (Bosterbio, USA) for 40 min at room temperature. The cells were then incubated with mouse anti-Nestin (Cell Signaling Technology, USA), rabbit anti-Pax6 (Cell Signaling Technology, USA), and rabbit anti-Sox2 (Cell Signaling Technology, USA) overnight at 4°C. On the next day, cells were washed with PBS containing 0.1% Tween 20 (PBST) (BBI Life Science, Shanghai, China) and incubated with Alexa Fluor-conjugated secondary antibodies (1000×, Invitrogen, USA) in PBST for one hour at room temperature (RT). Nuclei were stained with DAPI. Images were captured by a FV3000 confocal laser scanning microscopy (Olympus Optical Co. Ltd, Tokyo, Japan).

Hydrogels were collected at specified time points and
washed at room temperature with DPBS for 5 min. The washed hydrogels
were fixed with paraformaldehyde (4%) for 45 min at room temperature.
Following one wash with DPBS and two washes with DPBS with 1% bovine
serum albumin (BSA) and 0.3% Triton X-100 at 5 min each, the gels
were blocked and permeabilized with DPBS containing 1% BSA and 0.3%
Triton X-100. After blocking and permeabilization, the hydrogels were
incubated overnight at 4 °C with specified primary antibodies
(for neuroectoderm: 1:200 diluted rabbit anti-PAX6, Cell Signaling;
for non-neural ectoderm: 1:50 diluted mouse anti-AFP2α, Santa
Cruz Biotechnology; for mesoderm: 1:200 diluted rabbit anti-T, Cell
Signaling; for definitive endoderm: 1:200 diluted rabbit anti-SOX17,
Cell Signaling). After three additional washes with DPBS containing
1% BSA and 0.3% Triton X-100 for 45 min each at room temperature,
the gels were incubated with corresponding secondary antibodies (1:200
diluted goat anti-mouse AF647, Santa Cruz Biotechnology or 1:200 anti-rabbit
AF555, Cell Signaling) overnight at 4 °C. Three additional 30
min DPBS washes were conducted followed by 1 h counterstaining with
DAPI nuclear stain. After three final 10 min washes with DPBS, the
gels were imaged as described above.