Propylene oxide

Samples were incubated in propylene oxide (ProSciTech, 2×5 min) and embedded in resin. Embedding consisted of a 2 h incubation in 1:1 mix of propylene oxide and resin (Procure 812, ProSciTech); 10 min incubation at 60°C in resin, followed by overnight incubation at room temperature in the dark in resin; and finally embedding in resin at 60°C for at least 24 h to allow curing.

Undifferentiated mDPSC (3 × 10⁶) were liberated with trypsin from two T75 flasks and centrifuged at 200 × g for two minutes. Cells were resuspended in 1.5 mL 1.25% Gluteraldehyde (ProSciTech; Kirwan, Queensland, Australia) and 4% PFA (Sigma Aldrich; Sydney, New South Wales, Australia) (EM fixative) and stored at 4°C for 24 hour. All subsequent preparation was performed in a fume hood. Cells were washed with 4% sucrose in PBS for five minutes, then post fixed in 2% Osmium tetroxide (ProSciTech; Kirwan, Queensland, Australia) for 45 minutes. Cells were next dehydrated by serial incubations with 70%, 90%, 95% and 100% ethanol, then incubated in Propylene oxide (ProSciTech; Kirwan, Queensland, Australia) for 20 minutes. A 1:1 mixture of Propylene oxide:resin was applied for one hour then 100% resin, 2× one hour. Cells were then embedded in fresh 100% resin and polymerized at 70°C for 24 hours. The resin mixture was made of 10 mL Procure (ProSciTech; Kirwan, Queensland, Australia) 812, 6 mL Araldite (ProSciTech; Kirwan, Queensland, Australia) 502, 22 mL DDSA (ProSciTech; Kirwan, Queensland, Australia) and 560 μL DMP (ProSciTech; Kirwan, Queensland, Australia). Ultrathin sections were taken and imaged on a Philips CM100 TEM.