Visualizing GFP-labeled Organoids

GFP-labeled organoid was prepared by labeling day 35 organoids with a 1/500 dilution of human adenovirus type 5 expressing eGFP under control of a CMV promoter (Vector Biolabs; Ad-GFP). Three days after viral labeling, organoids were fixed and SHIELD-processed in supernatant of 2% P3PE solution, (as described above) but without clearing. Organoids were then embedded in low melt agarose and sliced at 200 µm thickness by vibratome (VT1000S, Leica Biosystems, Germany). Organoid slices were subject to passive clearing using the 0.2 M SDS, 50 mM phosphate (pH7.3) clearing buffer at 37C for 24 h and extensively washed by PBST. Fluorescence in situ hybridization-hybridization chain reaction (FISH-HCR) of GFP was performed as described before^{26 (link)} using 50nt GFP probes and B1 Alexa 647 hairpin. FISH-stained sample was imaged by an Olympus confocal microscope (FV1000MPE) with 20X 1.0 NA water objective (XLUMPlanFL N).