Liver tissue biopsies (n = 4) were collected during liver transplantation and fixed in 4% PFA for 24 h, followed by dehydration, paraffin embedding, and sectioning according to standard procedures. Automated multiplex immunofluorescent staining was further performed using the Ventana Benchmark Discovery (Ventana Medical Systems Inc.). For the novel series, following deparaffinization and heat-induced antigen retrieval with CC1 (#950-500, Ventana) for 64 min at 97 °C, the tissue samples were incubated with primary antibodies at 37 °C in a step-by-step manner. The antibody denature step was performed between every antibody incubation and visualization using CC2 (#950-123, Ventana) for 20 min at 100 °C. Keratin 19 (KRT19) was incubated for 32 min, detected with Universal HQ kit (#760-275, Ventana), and visualized with Red610 (#760-245, Ventana) for 4 min. TGF-β was incubated for 32 min, detected with Universal HQ kit, and visualized with R6G (#760-244, Ventana) for 4 min. N-cadherin was incubated for 32 min, detected with omnimap anti-mouse HRP, and visualized with FAM for 4 min. Slides were incubated in PBS with DAPI for 15 min and covered with an anti-fading medium (DAKO, S3023). Slides were scanned using the ZEISS Axio Imager 2.0.
Information on the antibodies used is listed in Table S3.
Shi S., Roest H.P., van den Bosch T.P., Bijvelds M.J., Boehnert M.U., de Jonge J., Dekker S.O., de Vries A.A., de Jonge H.R., Verstegen M.M, & van der Laan L.J. (2023). Modeling bile duct ischemia and reoxygenation injury in human cholangiocyte organoids for screening of novel cholangio-protective agents. eBioMedicine, 88, 104431.
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