Prolong antifade containing dapi

For immunofluorescence studies, cells were grown on glass coverslips, washed in PBS and fixed in 4% paraformaldehyde-PBS solution for 20 min at room temperature. Cells were then permeabilized for 10 min in PBS containing 0.2% Triton X-100, washed in PBS and then blocked for 30 min at room temperature with blocking buffer containing 10% goat serum in PBS. After blocking, slides were incubated overnight with 1:50 dilution of the corresponding primary antibody, washed extensively with blocking buffer and stained with the appropriate AlexaFluor-conjugated secondary antibody for 1 h at room temperature. Samples were washed extensively and mounted with Prolong antifade containing DAPI (Invitrogen). For double immunofluorescence staining, secondary antibodies were properly checked to avoid cross-reaction with isotype or species of the primary antibody immunoglobulin. Confocal images were acquired with a Leica TCS SP8 laser-scanning microscope (Leica, Germany). The series of images obtained from confocal z-stacks and orthogonal plane reconstruction were processed and analyzed using ImageJ software. Co-localization analysis was performed using the Colocalization Threshold plugin for ImageJ [32 (link)], which generates a merged image with white areas displaying the co-localization of two signals.

Both intracellular and extracellular MFG-E8 expression was measured in BAL cytospins using immunohistochemical analysis. For intracellular expression, cytospins were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.3% triton in PBS and blocked with 2% BSA in 0.3% triton. After blocking, cells were incubated with mouse anti-MFG-E8 (Santa Cruz Biotechnology, Dallas, TX USA) (1:150 dilution in blocking buffer) overnight at 4 °C. Cells were then washed and then incubated with goat anti-mouse (alexaflour 555, Invitrogen, Grand Island, NY, USA) (diluted 1:1000 in blocking buffer) for 1 h. For cell surface expression of MFG-E8, cytospins were fixed with 2% paraformaldehyde in PBS and blocked with 2% BSA in PBS (no triton was added) without permeabilization. After blocking, cells were incubated with mouse anti-MFG-E8 (1:150 dilution in blocking buffer without triton) at room temperature for 30 min. Cells were then washed and then incubated with goat anti-mouse alexaflour 555 (diluted 1:1000 in blocking buffer without triton) for 1 h. After incubation with the secondary antibody, cells were washed with PBS, air dried and then mounted with ProLong Antifade containing DAPI (Invitrogen, Grand Island, NY, USA). Images were captured using confocal microscopy (Zeiss LSM 700).

Formalin-fixed monolayers of NiV-infected cells grown in 8-well chamber slides were washed in DPBS prior to incubation in 0.2% Triton X-100 for 7 min, followed by blocking in 4% BSA/PBS for 10 min. Slides were then incubated with NiV-specific rabbit antisera, washed twice, then incubated with an anti-rabbit Alexa Fluor 488 antibody (Life Technologies). After washing twice, slides were mounted using ProLong Antifade containing DAPI (Invitrogen, Carlsbad, CA, USA) and visualized by confocal microscopy.

Females or males larvae at the 3^rd instar wandering stage were dissected in PBS for separation of the eyes imaginal discs. The separated tissue was fixed for 20 min in fixation solution (4% paraformaldehyde in PBS) and washed 3 times in PBT (0.3% Triton in PBS). The tissues were then blocked for 1 h in blocking solution (5% BSA in PBT) and incubated over night at 4°C in 200 µl of the primary antibody diluted 1∶50 in blocking solution and containing 1∶100 red phalloidin (Life Technologies). The samples were washed 3 times for 10 min each in PBT, incubated for 2 h in the secondary antibody 1∶200 and washed 3 times for 10 min in PBT. After being thoroughly washed with PBS, cells were mounted using ProLong Antifade containing DAPI (Invitrogen). Images were taken with LSM510 confocal microscope (Zeiss).

Melanoma cells were seeded at 3x10⁴ cells per well in 12-well plates on sterile cover slips. 24h post seeding attached cells were fixed and probed with primary antibody (S2 Table) and AlexaFluor488 conjugated goat-anti-mouse secondary antibody (Life Technologies, Mulgrave, Australia, 1:3000) and mounted with Prolong Antifade containing DAPI (Invitrogen, Carlsbad, USA).