Ix83 inverted fluorescent microscope

Immunofluorescence staining was performed according to our published method (Fan et al., 2010 (link)). The primary antibodies against PLD, YFP, BBS3, BBS1, and BBS5 and the secondary antibodies – Alexa-Fluor594-conjugated goat anti-rabbit IgG and Alexa-Fluor488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) – are listed in the key resources table with their suggested dilutions for immunofluorescence staining. Images were captured with an IX83 inverted fluorescent microscope (Olympus) equipped with a back illuminated scientific CMOS camera (Prime 95B, Photometrics) at 100× amplification and processed with CellSens Dimension (version 2.1, Olympus).

Mouse hearts were fixed in 10% phosphate-buffered formaldehyde (pH 7.4), paraffin embedded, and sectioned longitudinally at 5 μm thickness. Cardiac tissue sections were stained with hematoxylin and eosin (H&E) to detect changes in the morphology of the heart. Wheat germ agglutinin (WGA) staining was performed to assess cardiomyocyte cross-sectional area. Tissue sections were stained with WGA (1:100 dilution) following deparaffinization and antigen retrieval. After staining, slides were imaged with an Olympus IX83 inverted fluorescent microscope. Cross-sectional areas of around 65 myocytes per mouse heart were analyzed using ImageJ software to determine cellular hypertrophy.

Flavivirus stocks were titered by focus forming assay as previously described with minor modifications (Payne et al., 2006 (link)). Briefly, Vero cells were incubated with serial dilutions of virus for 48 hours. Cells were fixed in 4% paraformaldehyde (PFA; Electron Microscopy Sciences) diluted in phosphate buffered saline (PBS; Corning, #21-040-CM) for 10 minutes at room temperature (RT), permeabilized in ice-cold methanol for 5 minutes at RT, and washed in PBS. Monolayers were incubated with α-flavivirus E-protein monoclonal antibody (clone 4G2, a gift from Margaret Kielian, Albert Einstein College of Medicine) for 1 hour at RT, washed with PBS, and incubated with secondary α-mouse antibody conjugated to Alexa Fluor 488 (Invitrogen, A11029) for 30 minutes at RT followed by a 5-minute incubation with 300 nM 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) diluted in PBS. Foci were counted using an Olympus IX83 inverted fluorescent microscope.

Both Adherent monolayer cells and trypinised cells in suspension. After the HAL based treatment with either DMSO or DFO, cells were stained with 0.5 µM nuclear red for 15 min. Fluorescence was excited at 622 nm and emitted at 645 nm with a Cy5 optical channel for cellular imaging. Cy5 and PpIX imaging were performed simultaneously under the custom made IX83 inverted fluorescent microscope (Olympus, Japan). Nuclear red is here added for the purpose of localizing cells that may not display HAL-induced PpIX fluorescence.

U2OS cells (40,000 cells/well) were reverse transfected with FlavER plasmid using Fugene HD (Promega) in an 8-well chamber slide (Celltreat). Twenty-four hours post transfection, cells were fixed in 4% PFA in PBS, permeabilized with 0.1% Triton X-100 (Fisher) diluted in PBS, washed with PBS, and incubated with mouse α -V5 epitope tag monoclonal antibody (Invitrogen, 46-0705) or anti-dsRNA monoclonal antibody (Kerafast) for 1 hour at RT. The monolayers were washed 3X with PBS for 5 minutes each and probed with goat α -mouse conjugated to Alexa Fluor-647 (Invitrogen, A21236) for 30 minutes at RT. The cells were washed 3X with PBS for 5 minutes each and incubated in PBS containing 300 nM DAPI for 5 minutes at RT. The slide was mounted using Vectashield Antifade Mounting Media (Vector Laboratories) and a 24x50mm Premium Superslip (FisherScientific). Three fields of view per well were captured using an Olympus IX83 inverted fluorescent microscope.

Images of PpIX fluorescence were obtained using a custom made IX83 inverted fluorescent microscope (Olympus, Japan) equipped with a specially designed filter cube (Chroma). PpIX fluorescence was excited at 405 nm and emitted at 600 nm using appropriate LED lamps and a custom PpIX optical channel long pass filters. The fluorescence intensity was measured using Image-Pro Premier software (Media Cybernetics Inc., Rockville, MD 20852, USA). For cellular fluorescence measurement, background noise was removed by intensity threshold adjustment in image pro software. Using consistent detector system settings, objects of interest (PpIX fluorescent cells) that were distinguishable from the background were counted. The average intensity of each object was calculated over all pixels contained within the object contour, using a built-in software algorithm. The mean and standard deviation values were calculated by one-way ANOVA and standard variations test for each condition and each cell line (Minitab 18). The average fluorescence intensities of each cell in triplicate wells (n = 3) were recorded and were used to construct a normalised histogram. The histogram represents the average statistical distribution of cell fluorescence intensity data in each sample.