Dragonfly spinning disc confocal

Samples were fixed in 2–4% PFA, then washed and stored in PBS at 4 °C until needed. Immunofluorescent staining of organoids^{18 (link)} and podocytes^{29 (link)} was performed as previously described. Haematoxylin and eosin (H&E) staining was performed on organoid serial sections cut with a microtome at a thickness of 4 µm. Immunofluorescence was visualised with an LSM780 confocal microscope (Carl Zeiss) or a Dragonfly Spinning Disc Confocal (Andor Technology). 3D modelling software (Imaris 8, Bitplane, Connecticut) was utilised to reconstruct serial Z-stack images acquired via confocal microscopy.

PLA was performed using the Duolink in situ PLA Detection Kit (Sigma). Briefly, human breast cancer tissue or xenograft sections were dewaxed, hydrated and antigen repaired. After washed in PBS for 3 times, sections were incubated with Duolink blocking solution for 60 min at 37 °C. Primary mouse anti-human NANOG (1:100, cat# 4893, Cell Signaling Technology) and rabbit anti-human TAZ (1:100, cat# 83669, Cell Signaling Technology) antibodies in blocking solution was incubated for overnight at 4 °C. The PLA Probe incubation, ligation, and amplification reactions were performed according to the manufacturer’s instructions. Then, tissues were washed in Wash Buffer B and incubated with goat anti-human CK antibody (1:200, cat# ab219271, Abcam) for 1 h at RT, followed by incubated with Alexa Fluor 488-conjugated donkey anti-goat IgG (1:300, cat# A-11055, Thermo Fisher Scientific) for 1 h at RT. Finally, sections were mounted in Duolink in situ mounting medium with DAPI. Images were taken as z-stacks with a 0.25 μm step size by a Dragonfly Spinning Disc Confocal (Andor Technology). Max projections of z-stack were processed and analyzed by Imaris software.

Hoxb7-EGFP embryos (Srinivas et al., 1999 (link)) were isolated at E12.5 and cultured on the lower surface of a transwell membrane insert (Costar) in a glass-bottom dish (Mattek) in DMEM media (Sigma) containing 10% FCS (Sigma). Distance between the insert and coverslip was set at ~70 μm, controlled by a custom metal spacer between the rim of the dish, supplemented by 70 μm beads (Corpuscular Inc.), in a manner analogous to previously reported fixed-Z imaging approaches (Saarela et al., 2017 (link)). Samples were cultured and imaged at 10x magnification for >40 hr on a Dragonfly spinning disc confocal (Andor) in a stage-top incubator at 37 degrees C with 5% CO₂. 80 micron Z-stacks composed of 1.95 μm steps were taken every half an hour for GFP and bright field channels. Forming nephrons were identified from bright field images as epithelial structures.