Quantification of Virus RNA-Protein Colocalization

Cells were grown on glass coverslips in 24 wells plates. After infection at high MOI for the indicated times, cells were fixed with PBS-formaldehyde 4% (v/v) for 10 min, washed with PBS and permeabilized with PBS-BSA 1% (w/v)-Triton X-100 0.25% (v/v) for 10 min. Cells were incubated for 1 h in PBS-BSA 1% (w/v) with the appropriate primary antibodies, rinsed with PBS then incubated for 30 min with the appropriate Alexa Fluor-conjugated secondary antibodies and with Hoechst 33342 (1 μg/ml). Coverslips were rinsed in PBS, then mounted in ProLong diamond antifade reagent (Thermofisher). Cells were examined by confocal microscopy under a WLL Leica SP8 microscope except indicated otherwise. Representative pictures were taken.
Colocalization analysis were performed using the open-source software Icy (http://icy.bioimageanalysis.org/) [58 (link)]. We used Spot Detector plugin to automatically detect objects corresponding to RNP and vesicles (Rab11a or EEA1) and then Colocalization Studio plugin method: object based and statistical colocalization, SODA [35 (link)], to quantify the number of RNP at a distance less than 270 nm from the cellular vesicles.