An initial qualitative immunohistochemical screening of the expression of E‐cadherin, β‐catenin, cytokeratin 8 (CK8), vimentin and N‐cadherin was performed on formalin‐fixed paraffin‐embedded (FFPE) cell pellets of both non‐infected and persistently CDV‐infected DH82 cells (Supplementary material and Table S2 ).
Non‐infected DH82 and DH82Ond pi cells were seeded at a density of 0.03 * 106 cells/0.33 cm2 into 96 Microwell Nunc plates (Nunc GmbH & Co. KG, Thermo Scientific). All the immunostainings were performed in triplicates with negative controls in duplicates. 3 days after seeding, cells were fixed with 4% paraformaldehyde and immunofluorescence was performed according to a 2 days protocol with minor variations.25 To verify the persistent CDV infection state of DH82Ond pi cells, an immunolabelling with an anti‐CDV nucleoprotein (CDV‐NP) antibody (clone D110; kindly provided by Prof. Dr A. Zurbriggen, University of Bern, Switzerland) was performed as previously described.13 Furthermore, cells were immunolabelled for E‐cadherin, β‐catenin and cytokeratin 8 as epithelial markers, and for vimentin and N‐cadherin as mesenchymal markers. All details regarding the aforementioned antibodies are listed in Table 1 . For negative controls, the first antibody was replaced with rabbit serum, Balb/c ascitic fluid or goat serum, respectively, at corresponding protein concentrations. For all the aforementioned markers, the percentage of immunopositive cells was determined for each group (non‐infected DH82 cells and DH82Ond pi cells) by counting 5 evenly distributed fields per well, taking pictures at a 400× magnification using a fluorescence microscope (Olympus IX‐70, Olympus Optical Co. GmbH) equipped with an Olympus DP72 camera and Olympus cell sense standard software version 2.3. The analysed pictures were taken from areas of different confluence (low, medium, high) for each marker and for both persistently CDV‐infected and non‐infected DH82 cells. Besides the determination of the overall percentage of positive cells for each marker, the number of positive cells based on the cell shape (round, spindle or multinucleated giant cells) as well as the number of positive cells based on the intracellular localization (membranous, membranous to cytoplasmic, diffusely cytoplasmic, focally cytoplasmic) was additionally evaluated. Detailed descriptions of the cell shape and of the intracellular distribution pattern are available as supplementary material. Statistical analysis as well as graphical visualization was carried out using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com . The values were analysed with Student's t test, setting the significance level at P ≤ .05.
Non‐infected DH82 and DH82Ond pi cells were seeded at a density of 0.03 * 106 cells/0.33 cm2 into 96 Microwell Nunc plates (Nunc GmbH & Co. KG, Thermo Scientific). All the immunostainings were performed in triplicates with negative controls in duplicates. 3 days after seeding, cells were fixed with 4% paraformaldehyde and immunofluorescence was performed according to a 2 days protocol with minor variations.
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