780 upright confocal microscope

hiPSC-NPCs, hiPSC-neurons and NGN2-neurons were fixed in 4% paraformaldehyde in PBS at room temperature for 10 min. hiPSC-NPCs were permeabilized at room temperature for 15 min in 1.0% Trition in PBS. All cells were blocked in 5% donkey serum with 0.1% Triton at room temperature for 30 min. Primary antibodies, including rabbit anti-NESTIN (Millipore), 1:500; goat anti-SOX2 (Santa Cruz), 1:500; chicken anti-MAP2 (Abcam), 1:500–1:5,000; mouse anti-SYN1 (Millipore), 1:500; mouse anti-GFP (ThermoFisher), 1:1,000; rabbit anti-RFP (ThermoFisher), 1:1,000, rabbit anti-GABA (Sigma) 1:500; were incubated overnight at 4 °C. Secondary antibodies including Alexa donkey anti-rabbit 488, 568, 647 (Invitrogen), Alexa donkey anti-mouse 488, 568, 647 (Invitrogen) and Alexa donkey anti-chicken 647 (Invitrogen) were used at 1:500–1:1,000. DAPI (0.5 μg/mL) was used to visualize nuclei. Coverslips were mounted with Vectashield and imaged using the Zeiss upright 780 confocal microscope.

For histology analysis, 5-μm OCT-embedded cryosections were subjected to Fast Green/Sirius Red staining. For immunohistochemistry analysis, cryosections were subjected to antigen retrieval in preheated sodium citrate buffer (pH 6.0, 98°C) for 10 min with the PT Link system (Dako). For cryo-sectioned tissues, deparaffinizing and antigen retrieval procedure were replaced by treatment of 1% Triton X-100 in PBS for 10 min at room temperature. After pre-incubation with 10% non-immune goat serum (Thermo Fisher Scientific) to prevent non-specific binding, tissue sections were incubated with primary antibodies at 4°C overnight and subsequently with appropriate Alexa-Fluor conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature. Finally, sections were stained with DAPI (Sigma) and mounted in VECTASHIELD antifade mounting medium (Vector Laboratories). Images were captured with Olympus BX41 (Olympus) or Zeiss Upright 780 confocal microscope (Zeiss). Section information was pre-coded and blind to procedure performers including embedding, sectioning, staining and imaging.

For frozen tissue samples, frozen bladder tissue were sectioned and fixed in ice-cold acetone. After blocking with 1% BSA-PBS (Gibco), samples were stained with primary and secondary antibody. For whole mount bladders visualization, bladders were fixed for 2 h in 4 % PFA and blocked with buffer having 0.3 % Trion, 2.5 % normal goat serum (Gibco) in 1% BSA-PBS. Then, samples were stained with primary and secondary antibodies. For imaging human BEC, 5637 cells were grown on glass cover slip and treated as described. 4 % PFA fixed cells and 0.1 % saponin in 1% BSA-PBS solution permeabilized and blocked the samples. Primary antibody and secondary antibodies and phalloidin-Alexa647 (Invitrogen) stained samples. A Nikon ECLIPSE TE200 and Zeiss 780 upright confocal microscope were used for obtaining confocal images via a channel-series approach. For in vitro live imaging, 5637 BECs were grown on MatTek plate (MatTek Corporation) and were infected with UPEC or applied with granules as described. After propidium iodide (Molecular Probes) was applied to the media of cells, cells were placed under live cell station which is maintained at 37°C and 5% CO₂ with humidification. Zeiss Axio Observer microscope captured live moments.

Brain sections were blocked using 0.1% horse serum dissolved in 1% BSA in 0.3% Triton X-100 in PBS. Sections were reacted to polyclonal anti-ROBO4 antibody (Abcam, Cambridge, MA, USA). Antibodies were validated using a negative control method. Primary antibodies were specific for rat and used in 1:200 dilutions in 1% BSA in 0.3% Triton X-100 in PBS. Sections were then incubated with 1:1000 dilutions of Texas Red-conjugated goat anti-mouse or goat anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA) for 2 h. Samples were washed with PBS and imaged. ROI were imaged using a Zeiss 780 upright confocal microscope and were analysed, by a researcher blinded to grouping, for optical density using Image J software.