All embryos used for staining were harvested at either E12.5 or E13.5 embryonic stages, and heads were fixed in 4% paraformaldehyde (PFA) overnight at 4°C. After PFA fixation, samples were prepared in three ways. (1) For FFPE staining, after dehydration in ethanol and xylene, samples were embedded in paraffin, and frontal 10 µm sections were prepared. Followed by stepwise rehydration, antigen retrieval in 0.1 M citrate buffer, pH 6.0, under pressure boiling for 12 min was carried out. (2) For cryosection staining, after dehydration in 30% saccharose for 24 hr, samples were embedded in OCT, and 100 µm cryosections were obtained. (3) For whole-mount staining, whole heads were directly processed to permeabilization and blocking after PFA fixation.
Tissues were blocked in 5% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 for FFPE sections or with 0.5% Triton X-100 for thick cryosections or whole mounts for 1 hr and incubated in primary antibody solution (1% BSA in PBS and 0.1% Triton X-100 for 10 µm sections or 0.5% Triton X-100 for 100 µm sections and whole mounts). Primary antibody incubation was overnight for FFPE sections, two nights for cryosections, and three nights for whole-mount staining at 4°C. Primary antibodies used: SOX9 (Merck Sigma, AB5535), TRKA (R&D Systems, AF1056), MEIS2 (GeneScript, custom), TUJ1 (R&D Systems, MAB1195), EDAR (R&D Systems, AF745), Lef1 (Cell Signaling, C12A5), beta-galactosidase (Abcam, ab9361), SOX2 (R&D Systems, MAB2018), FOXD1 (Abcam, AB129324). Primary antibody solutions were washed out with PBS, Triton X-100 and incubated in fluorescent secondary antibody solutions for 1 hr for FFPE sections, 3 hr for cryosections at room temperature or overnight for whole mounts at 4°C. Secondary antibodies: donkey anti-mouse, -rabbit, -goat with Alexa Fluor 488, 568, or 647 fluorophores (Thermo Fisher Scientific). Primary and secondary antibodies were used at 1:500 for thick section and whole-mount staining and at 1:1000 for thin section staining. Immunofluorescent images were scanned by spinning disc confocal microscopy using an Olympus SpinSR10. Imaging was performed for whole-mount stainings after tissue clearing for 1 hr at room temperature. Acquired z-stacks were maximum intensity projected using ImageJ. Figures were assembled using Photoshop.
For cell proliferation assays, EdU was injected intraperitoneally to pregnant females at E12.5 for 2 or 18 hr before harvesting (0.5 ml per mouse, 2.5 mg/ml). EdU Base Click kit was used for EdU detection according to the manufacturer’s protocol on FFPE sections.
Tissues were blocked in 5% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 for FFPE sections or with 0.5% Triton X-100 for thick cryosections or whole mounts for 1 hr and incubated in primary antibody solution (1% BSA in PBS and 0.1% Triton X-100 for 10 µm sections or 0.5% Triton X-100 for 100 µm sections and whole mounts). Primary antibody incubation was overnight for FFPE sections, two nights for cryosections, and three nights for whole-mount staining at 4°C. Primary antibodies used: SOX9 (Merck Sigma, AB5535), TRKA (R&D Systems, AF1056), MEIS2 (GeneScript, custom), TUJ1 (R&D Systems, MAB1195), EDAR (R&D Systems, AF745), Lef1 (Cell Signaling, C12A5), beta-galactosidase (Abcam, ab9361), SOX2 (R&D Systems, MAB2018), FOXD1 (Abcam, AB129324). Primary antibody solutions were washed out with PBS, Triton X-100 and incubated in fluorescent secondary antibody solutions for 1 hr for FFPE sections, 3 hr for cryosections at room temperature or overnight for whole mounts at 4°C. Secondary antibodies: donkey anti-mouse, -rabbit, -goat with Alexa Fluor 488, 568, or 647 fluorophores (Thermo Fisher Scientific). Primary and secondary antibodies were used at 1:500 for thick section and whole-mount staining and at 1:1000 for thin section staining. Immunofluorescent images were scanned by spinning disc confocal microscopy using an Olympus SpinSR10. Imaging was performed for whole-mount stainings after tissue clearing for 1 hr at room temperature. Acquired z-stacks were maximum intensity projected using ImageJ. Figures were assembled using Photoshop.
For cell proliferation assays, EdU was injected intraperitoneally to pregnant females at E12.5 for 2 or 18 hr before harvesting (0.5 ml per mouse, 2.5 mg/ml). EdU Base Click kit was used for EdU detection according to the manufacturer’s protocol on FFPE sections.
