Organoid Embedding and (Immunohistochemistry) Staining

After individual treatment as described above, fixation of the organoids were employed by administration of the zinc formalin fixative Unifix (Klinipath, Duiven, The Netherlands) for 30 minutes. Next, Unifix was washed off and replaced with 70% ethanol before resuspension in heated liquid Histogel^TM (Thermo Fisher Scientific, Waltham, WA, USA). After storage at 4°C for 1 hour, the Histogel^TM was placed in 70% ethanol. Further FFPE (formalin-fixated paraffine-embedded) treatment was performed by the pathology department of UMC+ by dehydration of the organoids with subsequent paraffine embedding. After cutting, slides were obtained for further staining.

Haematoxylin and eosin (HE) staining was performed by staining with hemalaun followed by counterstaining with eosin. As ML contain iron oxides, Prussian blue staining was used for visualization of the ML. Prussian blue staining was conducted by the pathology department of RWTH Aachen University Hospital by staining with Berliner Blau (Merck KGaA, Darmstadt, Germany), counterstaining with Kernechtrot (Merck KGaA, Darmstadt, Germany; Waldeck GmbH & Co. KG, Muenster, Germany) and then dehydration in an ascending alcohol series.

For immunohistological analysis, we used the ZytoChem-Plus AP Polymer-Kit (Zytomed Systems GmbH, Bargteheide, Germany) according to the manufacturer’s directions. Sample slides were treated with the primary antibodies and then counterstained with hemalaun. Antibodies used for immunohistochemistry (IHC) staining were anti-CK (Cytokeratin) antibodies (Dako Denmark A/S, Glostrup, Denmark) for verification of cell differentiation, anti-Ki67 antibodies (Dako Denmark A/S, Glostrup, Denmark) for cell proliferation rate evaluation and anti-CC3 (Cleaved Caspase-3) antibodies (abcam, Cambridge, United Kingdom) for apoptosis detection. The TissueFAXS PLUS system (TissueGnostics, Vienna, Austria) was used for imaging and qualitative evaluation. Quantitative analysis of Ki67-expression and CC3-expression was performed with StrataQuest Analysis Software (TissueGnostics, Vienna, Austria).