Ab79351

For the histology study, the specimens were harvested, fixed in 10% neutral buffered formalin, decalcified, and then embedded in paraffin for preparation of serial sections (5 μm thick). The sections were stained with Hematoxylin-Eosin (H&E) and examined with light microscopy.
For immunohistochemistry of regenerated tooth, briefly, after the samples were fixed with 4% PFA, decalcified, dehydrated and embedded in paraffin, they were cut into 10 μm thick sections. Serial sections were permeabilized in 0.4% Triton X-100 and blocked in PBS containing 5% BSA. Sections were incubated with primary antibodies and overnight at 4°C, then washed and incubated for 1 h at 37°C with the respective secondary antibodies followed by Hematoxylin. The primary antibodies were as follows: anti-SOX2 (ab79351, Abcam, 1:200), anti-BMP4 (ab39973, Abcam, 1:100) and anti-WNT10b (ab66721, Abcam, 1:200). Slices were analyzed using a microscope (BX43 Olympus) with an attached Olympus DP72 digital camera system.

The following antibodies were used for western blotting: rabbit monoclonal anti-CD24 (1:500, catalog number ab179821, Abcam, MA, USA), rabbit monoclonal anti-CD44 (1:500, catalog number ab189524, Abcam), rabbit recombinant multiclonal anti-CD133 (1:800, catalog number ab278053, Abcam), mouse monoclonal anti-Nanog (1:1000, catalog number ab173368, Abcam), rabbit monoclonal anti-OCT4 (1:500, catalog number ab200834, Abcam), mouse monoclonal anti-SOX2 (1:1000, catalog number ab79351, Abcam), rabbit polyclonal anti-DLG5 (1:1000, catalog number ab231283, Abcam), rabbit polyclonal anti-PRDM16 (1:1000, catalog number ab106410, Abcam), mouse monoclonal anti-ErbB-2 (1:500, catalog number sc-33684, Santa Cruz Biotechnology, CA, USA), and mouse monoclonal anti-β-actin (1:1000, catalog number A5441, Sigma-Aldrich, MO, USA). Other primary antibodies and secondary antibodies and experimental procedures for immunoblotting are provided in the supplementary experimental procedures^{10 (link)}.

Cerebral organoid slices were fixed in 4% PFA for 20 minutes. Slices were blocked and permeabilized in 5% normal donkey serum 0.25% Triton X-100 for 1 hour at room temperature. Primary and secondary antibody mix was prepared in 5% normal donkey serum 0.1% Triton X-100 and incubated overnight at 4 °C. Samples were mounted in VECTASHIELD HardSet (Vector Laboratories) and mosaics (3 × 3 tiles) were acquired with CFI Apo LWD Lambda S ×40 objective (NA 1.15; WD 0.61–0.59, Nikon). Primary antibodies used included: sheep anti-EOMES (1:200, R&D Systems AF6166), chicken anti-GFP (1:500, Abcam Ab13970), mouse anti-Sox2 (1:500, Abcam Ab79351) and rabbit anti-NeuroD2 (1:500, Abcam Ab104430).

Antibodies for CTIP2 (ab18465, 1:500), CUX1 (ab54583, 1:500), PE-conjugated anti EGFR (ab231, 1:50), LGALS1 (ab25138, 1:1,000), Lin28 (ab46020, 1:1,000), PHH3 (ab5176, 1:250), PLZF (ab104854, 1:100), POU3F2 (ab94977, 1:1,000), SATB2 (ab51502, 1:50), SOX1 (1:1,000), SOX2 (ab79351, 1:500), TBR1 (ab31940, 1:200), TBR2 (ab23345, 1:200) were from Abcam. Antibodies for BrdU (347580, 50 μl per test), KI67 (556003, 1:1,000), phycoerythrin-conjugated (PE) SSEA-3 (560237, 20 μl per test), PE-conjugated F11R (552556; 20 μl per test), Alexa Fluor 647-conjugated TRA-1-60-647 (560850, 5 μl per test), Alexa Fluor 647-conjugated TUJ1 (560340, 1:500) were from BD Biosciences. Antibodies for DCX (AB2253, 1:5,000), O4 (MAB345, 1:25), RELN (MAB5364, 1:200), Tyrosine Hydroxylase (TH, AB152, 1:500) were purchased from Millipore. Antibodies for FABP7 (51010-1-AP, 1:100), S100B (15146-1-AP, 1:100) were from ProteinTech. Antibodies for AP2α (3B5 concentrated, 1:100) and PAX6 (supernatant, 1:16) were from DSHB. Antibody for NESTIN (MO15012, 1:500) was from Neuromics. Antibody for GFAP (Z0334, 1:2,000) was from DAKO. Antibody for β-3-Tubulin (PRB-435P, 1:1,000) was from Covance. Antibody for GLAST (ACSA-1; 130-095-822, 1:10) was from Miltenyi Biotec. Antibody for OTX2 (AF1979; 1:40) was from R&D. Antibody for FOXA2 (SC-6554; 1:100) was from Santa Cruz Biotechnology.

First, all pars plicata were isolated from the eyeballs of donors 7–12 (Table S1). A part of the isolated pars plicata was cut by the cross‐section method and cryosectioned. The remaining pars plicata were dissociated and used for fluorescence‐activated cell sorting (FACS) and subsequent experiments. The pars plicata cryosections were subjected to immunofluorescence labeling with the primary antibody mouse monoclonal SOX2 (ab79351, Abcam; 1:200 dilution) at 4°C overnight. Then, the sections were washed carefully and incubated with a Cy3‐labeled goat anti‐mouse IgG (H+L) secondary antibody (Beyotime, A0521) for 1 h. Images were captured by confocal microscopy (LSM 800; Zeiss, Germany).

The following antibodies were used at the appropriate dilutions: mouse anti-Map2 (M4403, Sigma), rabbit anti-Gfap (Z0334, DAKO), mouse anti-Tuj1 (MAB1195, R&D Systems), rabbit anti-s100-β (ab52642, Abcam), rabbit anti-Dcx (ab18723, Abcam), rabbit anti-Tbr2 (ab23345, Abcam), rabbit anti-phospho-Histone H3 (PHH3; Ser10; 06–570, Millipore), mouse anti-Sox2 (ab79351, Abcam), mouse anti-Nestin (MAB353, Millipore), rabbit anti-Ythdf2 (RN123PW, MBL), mouse anti-anti-β-actin (A1978, Sigma).

After treatment, the samples (cells cultured on glass coverslips and frozen mouse skin sections) were fixed in 4% paraformaldehyde for 20 min. The samples were incubated with primary antibodies at 4°C overnight and washed three times with phosphate-buffered saline (PBS). Next, the samples were stained with fluorescent secondary antibodies for 1 h at 37°C. Primary antibodies against the following were used: vimentin (10366-1-AP, 1 : 200, Proteintech), E-cadherin (20874-1-AP, 1 : 200, Proteintech), N-cadherin (22018-1-AP, 1 : 200, Proteintech), SOX2 (ab79351, 1 : 100, Abcam), and Slug (#9585, 1 : 1000, CST). The following secondary antibodies were used: FITC-labelled goat anti-rabbit IgG (F9887, 1 : 500, Sigma) and DyLight 594-labelled goat anti-mouse IgG (ab96881, 1 : 500, Abcam). The nuclei were then counterstained with DAPI (Abcam) before imaging. IF images were captured using fluorescence microscopy (Leica Microsystems, Germany).