Alexa 488 conjugated anti rat antibody

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.

In vivo angiogenesis experiments were performed as previously described by Malinda (2009) (link). A mixture of basement membrane matrix (ice-cold phenol red-free, reduced growth factor, Thermo Fisher Scientific) and conditioned medium (0.5 mL, 9:1 proportion) was injected subcutaneously into two-months-old C57Bl/6 wild-type mice (n = 4). Each mouse received two implants, totaling eight plugs per group. A buffered saline was included as a negative control during the assay. After 7 days, the mice were euthanized and the plugs were excised, photographed, and processed. Quantification of blood vessels was achieved using immunofluorescent visualization of blood vessels on frozen Matrigel sections and by measuring the amount of hemoglobin (Hb) contained in the plugs. Frozen Matrigel sections were stained with rat anti-mouse CD31 antibody (R&D system, 0.125 μg/mL in BSA/normal serum solution) followed by incubation with Alexa 488-conjugated anti-rat antibody (Molecular Probes, 10 μg/mL in PBS) as described previously by Ribeiro et al. (2019) (link). For quantitation of functional vessels formed, the plugs were homogenized in distilled water and centrifuged at 2,400 × g for 5 min. The supernatant was mixed with Drabkin’s reagent (Sigma-Aldrich) for measurement of Hb. After 15 min at room temperature, the absorbance of the mixture was measured at 540 nm.