Pdgfra

Organoid spheroids were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, cat#15710), subjected to a sequential gradient of sucrose, and embedded in 20% sucrose/OCT at ratio 2:1 (Tissue-Plus, ThermoFisher Scientific, cat#4585). 5 μm cryosections were rehydrated and blocked with 5% normal donkey serum (Jackson ImmunoResearch Laboratories, cat#017-000-121) in PBS supplemented with 0.1% Triton X-100 (IBI Scientific, cat#IB07100). Slides were immunostained with primary antibodies in 3% BSA (Fraction V, Gibco, cat#15260-037) followed by appropriate Alexa Fluor secondary antibodies (Invitrogen) and mounted using Prolong Gold with DAPI (Invitrogen, cat#P36935). Samples were imaged using a Nikon A1 High Sensitivity Confocal Microscope at the University of Michigan’s Microscopy Core and processed with Nikon Elements software. Primary antibodies: N-Cadherin (R&D, cat#AF6426, 1:1000); ACTA2 (R&D clone 1A4, cat#MAB1420-SP, 1:50); PDGFRA (BD Biosciences clone αR1, cat#556001, 1:200); Synaptopodin (Progen clone G1D4, cat#690094S, 1:80); NPHS1 (R&D, cat#AF4269, 1:500); TNFΑRSF1A (R&D clone 16803, cat#MAB225SP, 1:20); VCAM1 (Invitrogen clone 1.4C3, cat#MA5-11447, 1:50).

Flow cytometry analysis and sorting of lives cells was performed for GFP, VCAM1 (diluted 1:100, biotin conjugated Abcam ab7224) detected with APC-Streptavidin conjugated secondary (1:100, Biolegend), and PDGFRA (BD Biosciences, 556001) detected with PE/Cy7 conjugated secondary (Biolegend, 405315), as described previously^{16 (link),18 (link),64 (link)}. Pluripotency markers used were ECAD (ThermoFisher Scientific, MA1-10192) detected with APC conjugated secondary (1 in 100), EpCAM-PE (Biolegend, 324205, diluted 1:100), CD9-FITC (BD Biosciences, 341646, diluted 1:100) and SSEA4-APC (Biolegend, 330418, diluted 1:100) were detected as For intracellular flow cytometry, cells were harvested with TrypLE Select, fixed in 4% paraformaldehyde for 15 min at room temperature, blocked and permeablised in block buffer consisting of 1 × Perm/Wash Buffer (BD) and 4% goat serum (Sigma) for 15 min at 4 °C. Cells were then incubated with ACTN2 antibody (Sigma, A7811, diluted 1:100) for 1 h at 4 °C and then Alexa Fluor 647 conjugated secondary (ThermoFisher Scientific, A-21235, diluted 1:1000) for 1 h at 4 °C. Collection of flow cytometric data was performed using BD Fortessa™ analyser and analyzed with FlowLogic software (Inivai Scientific). Cell sorting was done using FACS Diva™ and BD Influx™ cell sorters (BD Biosciences).