Embedding Organoids in Collagen Droplets

To embed organoids in collagen droplets, a sheet of Parafilm was soaked in 70 % ethanol, air dried, and pressed against the holes of a box of gel-loading pipette tips (1-200 μl, Fisher Scientific 02-707-138) to create a dimpled mold^{31 (link)}. One organoid was placed in each dimple and 30 μL of 7 mg/mL collagen I (Corning) was added. The droplets were incubated 25 minutes at 37°C and carefully resuspended in 3 ml of RB media in an untreated 6-well plate. The media was changed weekly and the droplets were imaged after 2 weeks of incubation using a Nikon Ti Inverted Widefield microscope and a Nikon 1 J1 Camera. Droplet diameters were measured using NIS Elements imaging software (Nikon) and normalized to the diameter of empty droplets from the same set, for a total of three sets. Droplets that failed to undergo compaction (∼ 20 % in control and ∼ 50 % in PKD samples) were excluded. Droplets were fixed with 4% paraformaldehyde for 20 minutes at room temperature, incubated 16 hours in 30 % sucrose (Sigma) in water, mounted in Tissue-Tek (Sakura), flash frozen, and cryosectioned onto SuperfrostPlus slides (Fisher). Sections were stained in Picro-sirius red solution (Sky-Tek laboratories) for one hour, rinsed in two changes of 0.5% acetic acid solution, and dehydrated in two changes of absolute ethanol before mounting. Immunofluorescence was performed as described below.