Ix81 cellr microscope

Frozen tissue sections or cultured liver cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) for 60 min. Sections were stained with primary antibodies overnight at 4 °C (see Supplementary Table S1 for complete primary antibody list). Sections were counterstained with DAPI (0.1 μg/ml, Sigma-Aldrich, St. Louis, MO) and mounted with fluorescent mounting medium (DAKO, Glostrup, Denmark). Images were captured with Olympus IX81 CellR microscope (Olympus Corporation, Hamburg, Germany) equipped with Hamamatsu Orca ER (Hamamatsu Photonics, Herrsching am Ammersee, Germany) camera and 10x or 40x objective and processed using Hokawo 2.1 software (Hamamatsu Photonics).

Mouse ovaries were frozen in Tissue Tek O.C.T compound (Sakura Finetek, Alphen aan den Rijn, Netherlands). Seven µm cryosections were cut and mounted on Superfrost Plus adhesion slides (Knittel Glass, Braunschweig, Germany). The tissue sections were fixed with 4% paraformaldehyde, followed by permeabilization with 0.1% Triton X-100. Blocking was performed with 4% normal goat serum (NGS). Ahr antibody (SA-210) in blocking buffer was added to the sections for overnight incubation followed by incubation with secondary antibody Alexa Fluor® 488 goat anti-rabbit IgG. Sections were counterstained with 1 µg/mL DAPI (Sigma-Aldrich, Munich, Germany) and mounted with Fluorsave Reagent (Calbiochem, San Diego, CA, USA). Visualization was performed with Olympus IX81 CellR microscope (Olympus Corporation, Tokyo, Japan).

Following in situ hybridization, larvae at stages 37–44 were dissected at the level of the jaw joint to enable whole mount visualization of teeth that had developed on the roof and floor of the mouth. Additionally, mouth roof epithelia of embryos hybridized with the Shh probe were excised and photographed as flat-mounts to prevent masking of the tooth-specific signal from the signal in the overlying neural tube. Whole mounts processed through in situ hybridization, skeletal stainings, and transplantation of GFP-labeled oral ectoderm were photographed as Z-stacks using motorized dissection microscopes (Olympus SZX12, Zeiss SteREO Lumar.V12). The final deep-focus images were acquired by merging the Z-stacks using maximum projection function. Larvae processed through transplantation of GFP-labeled oral ectoderm were further sectioned using the CM3050 cryomicrotome (Leica) and individual tooth germs were visualized using an anti-calbindin antibody (Sigma) as previously described (Barlow and Northcutt, 1997 (link); Soukup et al., 2008 (link)). Z-stacks of sections were taken on the Olympus Cell^R IX81 microscope and merged as maximum projection images.