186 Chapter 6 Organoid imaging and immunofluorescence For brightfield imaging, organoids were imaged using an AxioObserver Z1 compound microscope (Carl Zeiss), 2.5× and 5× objectives and an AxioCam MRm3 CCD camera (Carl Zeiss). For immunofluorescence images, organoids were processed following the previously published protocol53. After collection with ice-cold Ad. DMEM/F12 the organoids were kept on ice for 10 minutes to ensure BME degradation. After centrifugation at 80G at 4°C, the organoids were reconstituted in 4% PFA and kept at 4C for 45 minutes. After fixation, organoids were permeabilized using PBS containing 0.1% Tween 20 and kept in that buffer for 2 days. Primary and secondary antibody incubations were performed in PBS containing 0.1% Tween 20 and 0.2% BSA. Primary and secondary antibodies are listed on supplementary table 2. Organoids were imaged using an ImageXpress Micro Confocal High-Content Imaging System (Molecular devices) with a 20X waterimmersion objective (molecular devices). 50 slices with 2µm step size were acquired for each wavelength. Image processing was performed using Fiji v1.8.0. RNA isolation, reverse transcription and quantitative realtime qPCR RNA was isolated from the organoids using RNase easy Kit (Qiagen) as described by the manufacturer. NanoDrop (NanoDrop Technologies) was used to assess the quality and yield of total RNA. M-MLV Reverse Transcriptase (200U/µl) (Invitrogen) was used to perform reverse transcription as established by the manufacturer. qPCR was performed in 384 well format in duplicates (5 -10 ng per well) using FastStart Universal SYBR Green Master (Rox) (Sigmal Aldrich) using QuantStudio 7 Flex (Thermo Fischer Scientific). All primer (Integrated DNA technologies Inc) sequences are listed in supplementary table 1. β-actin served as endogenous control, and was used for normalization. Hepatocyte functional assays For albumin measurements, the supernatant was collected after 24 hours. Levels of albumin in the supernatant were measured with the human albumin ELISA kit (Abcam) as described by the manufacturer using the Synergy H4 Hybrid Microplate Reader (BioTek Instruments Inc). Urea secretion was assessed in the organoids supernatant using the QuantiChrom™ Urea kit as described by the manufacturer.
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