Immunofluorescence assays were performed as described previously (Krtková et al., 2016 (link)), To detect triple hemagglutinin (3HA) tag, anti-HA rat monoclonal antibodies 3F10 (Roche) diluted to 1:125 followed by Alexa 488-conjugated anti-rat antibody (Molecular Probes) diluted to 1:250 were used. To detect HALO tag 0.5 μM Janelia Fluor 549 (Promega) dye or HaloTag® TMR Ligand (Promega) were used. CWP1 was detected with Alexa 647-conjugated anti-CWP1 antibody (Waterborne, New Orleans, LA, United States).
Fluorescent images were acquired on a DeltaVision Elite microscope using a 100×, 1.4-numerical aperture objective and a PCO Edge sCMOS camera. Deconvolution was performed with SoftWorx (API, Issaquah, WA, United States) and images were analyzed using Fiji, ImageJ (Schindelin et al., 2012 (link)). Pearson Coefficient, Manders Correlation Coefficient and Costes’ automatic thresholding analyses were obtained using the JACoP plugin for ImageJ (Bolte and Cordelières, 2006 (link)). 3D viewing and manual scoring of cells were performed using Imaris (Bitplane, version 8.9). Figures were assembled using either Adobe Photoshop or Adobe Illustrator. A minimum of 120 cells were imaged for each cell line and timepoint post induction of encystation which corresponded to between 15 and 20 cells at each of our defined stages.
Fluorescent images were acquired on a DeltaVision Elite microscope using a 100×, 1.4-numerical aperture objective and a PCO Edge sCMOS camera. Deconvolution was performed with SoftWorx (API, Issaquah, WA, United States) and images were analyzed using Fiji, ImageJ (Schindelin et al., 2012 (link)). Pearson Coefficient, Manders Correlation Coefficient and Costes’ automatic thresholding analyses were obtained using the JACoP plugin for ImageJ (Bolte and Cordelières, 2006 (link)). 3D viewing and manual scoring of cells were performed using Imaris (Bitplane, version 8.9). Figures were assembled using either Adobe Photoshop or Adobe Illustrator. A minimum of 120 cells were imaged for each cell line and timepoint post induction of encystation which corresponded to between 15 and 20 cells at each of our defined stages.
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