Immunostaining of Urothelial Cells

USCs or differentiated urothelia were fixed overnight in 10% formaldehyde at 4 °C. After washing in PBS, the fixed samples were pre-embedded into 2% agar, cut vertically, put in transwells side face up, embedded again in paraffin blocks and sectioned. The slides were stained for H&E and immunostained for selected antibodies. For immunofluorescence staining, slides were deparaffinized, hydrated, blocked with 10% heat-inactivated horse serum (HIHS) and 0.3% Triton X-100 in PBS, incubated with primary antibody in 1% HIHS and PBS overnight at 4 °C and secondary antibody in PBS for 30–60 min at room temperature^{6 (link)}. The primary antibodies used were mouse monoclonal anti-keratin 20 (Abcam, ab854, 1:200), goat polyclonal anti-E-cadherin (R&D Systems, AF748, 1:500), goat polyclonal anti-uroplakin 3a (Santa Cruz, sc-15186, 1:500), mouse monoclonal anti-uroplakin 3a (Fitzgerald, 10R-U103a, 1:50), rabbit polyclonal anti-p63 (GeneTex, GTX102425, 1:1,000), rabbit monoclonal anti-K5 (Abcam, ab150074, 1:100) and mouse monoclonal anti-keratin 14 (Santa Cruz, sc-53253, 1:50). Alexa Fluor secondary antibodies and DAPI were used at 1:1,000 dilution. Further antibody information is provided in Supplementary Table 3. Samples were visualized on a Zeiss Axio Imager M2 Plus wide-field fluorescence microscope.