Oil immersion objective

For histological analysis, the cochleas were fixed by an intra-labyrinthine infusion with a fixative solution containing 3% glutaraldehyde, 2% formaldehyde, 1% acrolein and 2.5% dimethyl sulfoxide (DMSO) in a 0.08 M sodium cacodylate buffer. The cochleas were then decalcified, post-fixated and embedded in Spurr’s low-viscosity resin. Staining was performed using 1% methylene blue, 1% azur B and 1% borax in distilled water. The cochleas were subsequently divided into two halves along a standardized midmodiolar plane, then re-embedded in fresh resin, and cut to obtain semithin (1 μm) sections. More details about this procedure are reported in Kroon et al. (2017) [39 (link)]. The images were acquired by using a Leica DC300F digital camera mounted on a Leica DMRA light microscope with a 40× oil immersion objective (Leica Microsystems GmbH, Wetzlar, Germany).

A549 cells were plated on poly-d-lysine-coated 35-mm culture dishes (MatTek, Ashland, MA) and either treated with IFN-β (10 ng/ml) for 16 h or infected with DENV-2 (MOI, 3) for 48 h. The cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% or 0.5% Triton X-100, followed by blocking with 5% bovine serum albumin (BSA) for 30 min. Primary antibodies were diluted in 10% normal goat serum and incubated at room temperature for 2 h, followed by three washes in PBS and staining with fluorescently labeled secondary antibody (1:500) and nuclear DAPI (4′,6-diamidino-2-phenylindole) stain (Life Technologies). Primary labeled antibodies were diluted in 10% normal goat serum and incubated at room temperature for 2 h, followed by three washes in PBS, and postfixed with 4% paraformaldehyde in PBS. Images were collected on a Leica SP8 inverted confocal microscope with a 63× oil immersion objective (Leica Microsystems, Buffalo Grove, IL). Colocalization analysis was performed using Imaris software (Bitplane Inc., South Windsor, CT). FRET-by-FLIM analysis was performed as previously described (73 (link)).