Once complete, the MBAs were prepared for immunofluorescence imaging. The volume of the MBA was exchanged within the device by slowly manually injecting the solution with a 1-ml syringe with a 22-gauge blunt needle through the cell seeding inlet to the cell seeding outlet. Cells were fixed in a two-stage process and carried out under chilled PBS. Initial fixation was carried out using 2% (weight) paraformaldehyde in PBS for 10 min. After three brief rinses with PBS, cells were then exposed to absolute ethanol for 15 min, followed by three more brief rinses with PBS.

Blocking buffer was prepared with 10% donkey serum (Sigma-Aldrich) and 0.3% Triton X-100 (Sigma-Aldrich) in PBS. Rinse buffer consisted of 1% donkey serum and 0.05% Tween 20 (Sigma-Aldrich) in PBS. Devices were blocked with blocking buffer for 1 hour at room temperature, followed by a rinse with the rinse buffer. Primary antibodies of GATA3 (R&D Systems), WT1 (Santa Cruz Biotechnology), ECAD (BD Biosciences), and/or PAX2 (Life Technologies) were all diluted at 1:200 in rinse buffer and allowed to adsorb overnight at 4°C. The devices were then rinsed three times with rinse buffer. Donkey (H+L) secondary antibodies of AF488 anti-goat, AF568 anti-rabbit, and AF647 anti-mouse (Life Technologies) at a dilution of 1:500 with Hoechst 33342 (Life Technologies) were diluted 1:1000 in rinse buffer and allowed to adsorb for 1.5 hours at room temperature. Devices were lastly rinsed three times with rinse buffer, followed by a single time with PBS.

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