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.
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.
