Goat anti mouse conjugated to alexafluor488

Viral titers were determined using immunofluorescence on subconfluent MDCK II-MHC-II cells pretreated with PBS containing 0.2% BSA (Sigma-Aldrich) and 1.5 mg mL⁻¹ DEAE-dextran. At 48 hpi, the inoculum was removed, cells were fixed with 4% PFA (EMD), and the cell membrane was permeabilized with PBS containing 0.5% Triton X-100 (Sigma-Aldrich). Infected cells were stained with a polyclonal rabbit anti-H18 (in-house made, 1:500) and mouse anti-NP (in-house made, 1:500) in PBS containing 5% NGS (Vector Laboratories, Inc.). Primary antibodies were detected using secondary goat anti-rabbit (Jackson ImmunoResearch, 1:500) conjugated to Cy3 and goat anti-mouse conjugated to AlexaFluor488 (Jackson ImmunoResearch, 1:500) in PBS containing 5% NGS (Vector Laboratories, Inc.).

DRG explants were fixed in 4% paraformaldehyde solution in PBS for 15 min, washed once in PBS and blocked in 5% milk in Tris-borate buffer and 0.3% Triton X-100 for 1 h at room temperature (RT). Explants were incubated overnight at 4°C with mouse monoclonal antibody against βIII-tubulin (Millipore, MAB5564) diluted 1:10,000 in blocking solution. DRGs were washed twice in PBS and then incubated with goat anti-mouse conjugated to Alexa Fluor 488 (Jackson ImmunoResearch, 115-545-003) diluted 1:5000 in blocking solution for a minimum of 3 h at RT. Explants were imaged using a Zeiss ObserverZ.1 inverted epifluorescence microscope with an automated motorized stage (5× magnification with tilling). From a stitched master image of the plate generated by Zen 2 software (Zeiss), quarter DRG fields were cropped to generate a set of images for analysis using the R script program Axoquant 2.0 (Johnstone et al., 2018 (link)). Final measurements were plotted as the mean axonal area of DRGs from three embryos. Increments of 500 μm were used for statistical analysis (normalized to same increments in control condition).