For paraffin embedding, 3D thick layer cultures were fixed with 4% PFA at room temperature overnight. The PFA-fixed Matrigel was then transferred to a plastic Tissue-Tek Cryomold (Sakura Finetek, Torrance, CA, USA), preloaded with 60°C liquefied HistoGel (Thermo Scientific). After positioning the fixed 3D Matrigel at the center, the whole Cryomold with Histogel/Matrigel was transferred on ice and then incubated for 15 minutes until the Histogel was solidified. The Histogel/Matrigel complex was then further fixed with 4% PFA at 4°C overnight, washed 5 times with PBS, and sent for paraffin embedding (MGH pathology core, Charlestown, MA, USA). The paraffin blocks were then cut into 10 μm sections (Leica SM2010R sliding microtome, Leica Microsystems Inc., Buffalo Grove, IL, USA), mounted on polylysine-coated glass slides (Thermo Scientific), then incubated at 45°C overnight. The sections were deparaffinized by 2 changes of xylene for 5 minutes each and then serially transferred to 100%, 90%, 70% ethanol solution for 1 minute each. The sections were then rinsed with distilled water for 5 min. For IF and IHC, the antigen retrieval was performed by heating the slides for 30 minutes in Citrate-EDTA Buffer containing 10 mM citric acid (pH 6.2), 2 mM EDTA and 0.05% Tween-20.
