A1 resonant confocal microscope

Z-stacks from tissue sections of GlyT2-eGFP and GlyT2-Cre mice were obtained on a Nikon A1 Resonant Confocal microscope (Nikon Instruments Inc., Melville, NY) equipped with NIS-Elements High Content Analysis software (version 5.02, Nikon Instruments Inc., Melville, NY). Tissue sections containing labeled CeA and PnC neurons were first examined on a single Z-plane with the × 10 objective to survey the tissue section. Using a × 60 objective, an area (212.56 μm width × 212.56 μm height) within CeA and PnC sections was then sequentially scanned by the 488-, 561-, and 640-nm laser lines in 0.1 μm Z-steps throughout the 30-μm tissue section. Z-stacks were analyzed with NIS-Elements 5.0 Advanced Research software (version 5.02, Nikon Instruments Inc., Melville, NY). To visualize close appositions of CeA axons (labeled with mCherry) with GlyT2⁺ neurons (labeled with eGFP) in GlyT2-eGFP mice, a binary layer was configured to segregate putative synaptic contacts of > 50 nm in distance (due to technical limitations). These contacts were imaged in split-channels and orthogonal views. Then, z-stacks were reconstructed in three-dimension and volume was rendered.

GCaMP 3 (gift from Michael Laflamme Lab, Toronto)^{44 (link),47 (link)} and H9, CF01 iPSC were differentiated to cholangiocyte cysts and plated down in Lab-Tek Chamber Slide (Millipore) or in flow chamber (ibidi) 2 days prior to the assay. Day 27 hepatoblasts were aggregated and plated down 2 days prior to the assay. Chambers were pre-coated with fibronectin. The cells were replaced in Tyrode buffer before the assay. H9-derived-cholangiocytes and CF01 derived cholangiocytes were stained with 10 μM Fluo4 (Invitrogen) with 0.04% Pluronic F127 (Invitrogen) for 30 min prior to the assay. To measure calcium response to ATP and TUDCA (Sigma) in 3D cysts, GCaMP derived cholangiocyte 3D cysts were embedded in the type1 collagen gel just before the assay.
Calcium response to 20 μM ATP (Sigma) or flow (Perista BioMini Pump: ATTO) were analyzed using a confocal fluorescence microscope (NIKON A1 Resonant Confocal Microscope) and images captured using the Nikon Elements software.