Triton x 100

After three and 10 days of culture, Vk2 cells were fixed in 4% paraformaldehyde for 24 h and washed in phosphate-buffered saline (PBS; McMaster Immunology Research Centre Media Production Facility, Hamilton, ON, Canada). Cultures were then excised from transwell polystyrene inserts, stored in 70% ethanol, processed for histology and embedded in paraffin wax. Sections of 4–6 micron were cut and stained with hematoxylin and eosin (H&E) at the McMaster Immunology Research Centre Histology lab (Hamilton, ON, Canada). All samples were imaged on a Leica HC DMR microscope under 40× objective (Leica Microsystems, Wetzlar, Germany). To visualize the filamentous F-actin, after three and 10 days of culture, Vk2 cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (Mallinckrodt Inc., Paris, KY, USA), and blocked for 30 min in blocking solution (5% bovine serum albumin and 5% goat serum (Sigma-Aldrich) in 0.1% Triton X-100 (Mallinckrodt Inc.)). Cultures were incubated with Texas Red-conjugated phalloidin (Sigma Aldrich) for 1 h at 37 °C. Cultures were then washed three times with PBS, and the polystyrene inserts were excised and mounted onto glass slides. Samples were imaged using an inverted laser-scanning confocal microscope at the McMaster Electron Microscopy Facility at 40× objective (LSM 510, Carl Zeiss Canada Ltd., Toronto, ON, Canada).

Cells plated on 8-well glass chambers (Thermo Fisher) were fixed with 4% paraformaldehyde (Mallinckrodt Baker) for 15 minutes, permeabilized with 0.1% Triton X100 (Mallinckrodt Baker) for 10 minutes, and blocked with blocking buffer (5% BSA in PBS) for 1 hour at room temperature (RT). Cells were incubated overnight at 4°C with primary antibodies (Supplementary data, Table S2). On the next day, cells were incubated for 1 h at RT with secondary antibodies. Both primary and secondary antibodies were diluted in blocking buffer. Slides were mounted with a ProLong® Gold Antifade reagent with DAPI (Life Technologies), and confocal images were captured by using a LSM 510 Meta microscope (40x objective). Images were acquired using ZEN Black software. Counting of NKX2.1⁺ and PAX 6⁺ cells was performed by two independent raters in a blinded fashion. For each condition, at least three images with at least 100 cells per image were counted. The NKX2.1/PAX6 ratio was calculated based on cell counts for each condition. The experiments were conducted in triplicates. Purity of the hESC-derived MGE progenitor population was assessed by NKX2.1⁺ and PAX6⁺ cell count in ICC images. It was determined by the ratio of NKX2.1⁺ cells divided by the total number of generated NPC (NKX2.1⁺ and PAX6⁺ cells).

Eyelids from P3, P6, P8, P12 and P25 Arl13b-mCherry;Centrin2-GFP mice were dissected and fixed for 1 h in 4% PFA/1% Triton X-100 (Mallinckrodt Pharmaceuticals, Staines-upon-Thames, United Kingdom) in PBS. Eyelids were then processed for K14 staining on whole MGs or embedded in OCT. For whole MGs, prior to staining, most of the connective tissues and muscles covering the tarsal plate were removed and tarsal plates were permeabilized for 1 h with 2% BSA/1% Triton X-100/PBS at RT. MGs for whole mount samples and cryosections were stained with a rabbit polyclonal antibody directed to K14 (1:1000, 905301, BioLegend, San Diego, CA). Nuclei were counterstained with DAPI on cryosections. MG whole mounts (P3, P6, and P8) and cryosections (P12 and P25) were imaged with a Zeiss LSM880 confocal microscope. Serial optical sections were acquired in 1 µm steps. After 3D reconstruction of the z-stacks with Imaris, MGs were outlined using the Surfaces tool, and primary cilia and basal bodies were counted in MGs using the Spots tool. Centrin2-GFP-labeled centrioles were considered as a single basal body when less than 2 µm apart. Since the number of basal bodies was similar to the number of nuclei as shown in Supplement Fig. 4, the number of ciliated cells in MGs was determined by normalizing the number of primary cilia to the number of basal bodies and expressed as a percentage.