Richard allan scientific histogel specimen processing gel

At passage 4–5, NCC-MPCs were detached and dissociated into single cells using pre-warmed TrypLE™ Express, generating a solution of 1.6 × 10⁶ viable cells/ml. Then 5 μl droplets of cell solution were seeded in 12-well culture plate. Cells were incubated with serum-containing media for 2–4 h, which was then replaced with chondrogenic differentiation media (StemPro chondrogenic differentiation kit, Life Technologies) and refed every 2–3 days. Within 14 days, cells formed micro-masses that were stained using the Alcian Blue staining kit (Lifeline Cell Technologies), according to the manufacturer’s instructions. The harvested micro-masses were fixed with 4% PFA for 1 h, then embedded into liquefied Richard-Allan Scientific HistoGel Specimen Processing Gel (ThermoFisher Scientific), solidified on ice for 1 h, and finally paraffin embedded, sectioned, and stained with Alcian Blue stain. The stained micro-masses were visualized under light microscope for image capturing and analysis.

Organoid assay was performed as has been described previously.^{18 (link)} FACS-isolated basal cells, Sca-1⁺ luminal cell, eYFP⁺ Sca-1⁻ luminal cells, and eYFP⁻ Sca-1⁻ luminal cells from Pbsn-eYFP mice or FACS-isolated Sca-1⁻ and Sca-1⁺ luminal cells from WT-Sox2 and K8-Sox2 mice were cultured in Corning Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning, Tewksbury, Massachusetts) with advanced DMEM/F12 supplemented with B27 (Life Technologies, Grand Island, New York), 10 mM HEPES, glutamax (Life Technologies), penicillin/streptomycin, and the following growth factors: EGF 50 ng/mL (Peprotech, Rocky Hill, New Jersey), 500 ng/mL recombinant R-spondin1 (Peprotech), 100 ng/mL recombinant Noggin (Peprotech), 200 nM of TGF-β/Alk inhibitor A83–01 (Tocris, Ellisville, Missouri), and 10 μm Y-27632 (Tocris). Dihydrotestosterone (Sigma) was added to a final concentration of 1 nM.
To collect organoids for histological and IHC analyses, 1 mg/mL dispase (Invitrogen) was added and incubated for 1 hour at 37°C. Organoids were then fixed using 10% formalin for 10 minutes and resuspended in 50 μL of Richard-Allan Scientific HistoGel Specimen Processing Gel (Thermo Fisher Scientific) for preparation of paraffin embedded blocks.