Rabbit anti pax6

E13.5 dorsal telencephalon was microdissected, avoiding the lateral ganglionic eminence and structures ventral to it. The cortex was separated from the meninges, and cortices from samples of the same genotype were pooled and sliced into small, uniformly sized pieces. Tissues were digested with 2.5% Trypsin (Invitrogen, Carlsbad, CA), then dissociated into single cells by repeated pipetting. Cells were fixed in 4% paraformaldehyde (PFA), incubated with primary antibodies, and then secondary antibodies. Each step was carried out in 4°C for 30 mins, with rotation. RNAsin (NEB, Ipswich, MA) was added to buffers to prevent RNA degradation (Hrvatin et al., 2014 (link)). Cells were sorted using FACS Aria IIU (BD).
Antibodies: Rabbit anti-PAX6 (Biolegend 901301, 1:1000), Mouse anti-TUJ1 (Biolegend 801202, 1:1000)

Cerebral cortices were fixed in PFA overnight and sectioned coronally by Vibratome into 100 μm slices. Slices were washed in PBS and then permeated in PBST solution (0.2% Triton X-100 in PBS) for 15 min. Antigen retrieval was done with citrate buffer (100 mM citrate with 0.1% Triton X-100, pH = 6) in boiling water for 10 min. After cooling and PBS washing, slices were blocked at room temperature with 10% normal goat serum and 5% BSA in PBST solution. Primary antibodies were used with the following concentrations: mouse anti-Cntn1, 1:100 (LSBio); rabbit anti-Cntn1, 1:100 (Proteintech); mouse anti-Tuj1, 1:1000 (Convance); rabbit anti-NeuN, 1:500 (Millipore); rabbit anti-Pax6, 1:300 (BioLegend); rabbit anti-Tbr2, 1:500 (Abcam); mouse anti-RhoA, 1:100 (Santa Cruz). After incubation in primary antibody for two nights, slices were washed in PBS. Following the wash, secondary antibodies (Alexa Fluor, 1:500) were applied for 2 h at room temperature. Finally, the slices were counterstained with 0.5 μg/ml DAPI (Invitrogen) for 1 h. VECTASHIELD Mounting Media was added before sealing the slices. The slices were preserved and kept in a dark place.

For cryosections, embryos were fixed in 4% paraformaldehyde (PFA), cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) overnight at 4°C, embedded in OCT compound (Tissue Tec; Sakura Fine Technical, Japan), and sectioned at 10 μm using a cryostat (HM560; Thermo Fisher Scientific, Waltham, MA). Specimens were blocked with horse serum for 1 hr at room temperature and incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 hr at room temperature.
For flat-mount immunostaining of the RPE/choroid, the tissues were fixed with 4% PFA at room temperature for 30 min, and washed three times with PBS containing 0.5% Triton X-100 (PBST, Nacalai Tesque, Japan), incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 hr at room temperature (Zhu et al., 2012 (link)). The primary antibodies and dilutions used were as follows: goat anti-Aldh1a1 (1:1,000; Abcam), rabbit anti-Aldh1a3 (1:1,000; Sigma), rat anti-endomucin (1:400; Millipore), mouse anti-GS (1:1,000; Millipore), FITC-isolectin B4 (1:100; Vector Laboratories), rabbit anti-ZO-1 (1:100; Invitrogen), rabbit anti-GFP (1:1,000; Abcam), rabbit anti-ERG (1:400; Abcam), rabbit anti-Pax6 (1:200; BioLegend), and rabbit anti-Sox9 (1:200; Millipore).

Mouse anti-KBTBD8 antibodies (1 μg/mL in IB) were described previously (Werner et al., 2015 (link)). Mouse anti-Flag (F1804, clone M2, Sigma, 1:2000 in IB), rabbit anti-PAX6 (Biolegend, PRB-278B, 1:300 in IF), rabbit anti-CK2α (#2656S, Cell Signaling 1:500 in IB) rabbit anti-CK2β (A301-984, Bethyl,1:500 in IB), rabbit anti-CK2 substrate (#8738S, Cell Signaling 1:500 in IB), mouse anti-PCNA clone PC10 (sc56, Santa Cruz, 1:500 in IB), rabbit anti-GAPDH (#2118, clone 14C10, Cell Signaling, 1:10,000 in IB), rabbit anti-ARRB1/2 (#4674, clone D24H9, Cell Signaling, 1:1000 in IB), mouse anti-PKN1 (610687, clone 49/PRK1, BD Bioscience, 1:1000 in IB), rabbit anti-PP1 (#2582, Cell Signaling, 1:100 in IB), rabbit anti-PP2A A (#2041, clone 81G5, Cell Signaling, 1:1000 in IB), rabbit anti-PP2A B (#2290, clone 100C1, Cell Signaling, 1:1000 in IB), rabbit anti-PP2A C (#2259, clone 52F8, Cell Signaling, 1:1000 in IB), rabbit anti-CUL3 (Bethyl, 1:1000 in IB), rabbit anti-NANOG (#3580, Cell Signaling, 1:1000 in IB), goat anti-OCT4 (ac-8628, Santa Cruz, 1:1000 in IB), rabbit anti-SNAIL2 (#9585, clone C19G7, Cell Signaling, 1:500 in IB), rabbit anti-TCOF1 (11003–1-AP, Proteintech, 1:250 in IB), and rabbit anti-NOLC1 (11815–1 P, Proteintech, 1:1000 in IB) antibodies were commercially purchased.

Immunostaining of three germ layers was performed using a Human Three Germ Layer 3-Color Immunocytochemistry Kit (R&D Systems). For immunostaining of cells during cardiomyocyte differentiation, the pluripotent cells were grown and differentiated in a glass-bottomed 12-well plate (MatTek, MA), and then cells were fixed and permeated in the plate using a Human Cardiomyocyte Immunocytochemistry Kit (Thermo Fisher Scientific). The primary antibodies included mouse anti-TRA-1-60 (ESI BIO, ST11016), rabbit anti-Nanog (Stemgent, 09-0020), goat anti-Brachyury (Fisher Sciences, AF2085), rabbit anti-Pax6 (BioLegend, 901301), rabbit anti-Nkx2-5 and mouse anti-Tnnt2 (Thermo Fisher Scientific, A25973). Secondary antibodies included goat anti-mouse IgG (Alexa Fluor 488 conjugated, Thermo Fisher Scientific, A10680), donkey anti-goat IgG (DyLight 488 conjugated, Thermo Fisher Scientific, SA5-10086), goat anti-rabbit IgG (H + L) Alexa Fluor 488 (Thermo Fisher Scientific, A-11034), and donkey anti-rabbit IgG (Alexa Fluor 594 conjugated, Thermo Fisher Scientific, A21207). The images were taken using Leica DMi8 Microsystems and Zeiss LSM710 inverted confocal microscope, and then the images were processed using the Fiji software.

Antibodies were used at the following dilutions: rabbit anti-Pax6 (Biolegend, polyclonal, 1:300); Rat anti-CTIP2 (Abcam, clone 25B6, 1:300); Rat anti-tbr2 (Invitrogen, Eomes clone Dan11mag, 1:100); mouse anti-Satb2 (Abcam, clone SATBA4B10,1:50); Mouse anti-Neun (Millipore, clone A60, 1:200); and Rabbit anti-Tbr1 (Abcam, polyclonal, 1:100). Anti-Rabbit, Mouse, or Rat conjugated with Alexa Fluor 488 or Alexa Fluor 647 fluorophore secondary antibodies (Life Technologies) were used at 1:500 in PBTA solution.