Lasx 2020

Immunolocalization studies of Cy3-labeled dsSelK uptake were performed on ISE6 cells grown on glass cover slides. After transfection with Cy3-labeled dsRNA, ISE6 cells were fixed in 1× PBS containing 4% para-formaldehyde. The cells were permeabilized using 0.1% Triton X-100 (Sigma Aldrich) for 15 min. Cells were then incubated with the fluorescent dye Alexa Fluor phalloidin (Invitrogen) in 1× PBS for 1 h in the dark. All incubations and washes were performed on a rocking plate at room temperature unless otherwise indicated. Cover slides were mounted on glass slides using a VECTASHIELD antifade reagent with a DAPI (Vector Laboratories) mounting medium. Cells prepared in this manner were mounted and viewed under a Leica TCS SP8 LIGHTNING confocal microscope ran with LASX 2020 software (Leica, Wetzlar, Germany) using a 40× objective.

Delivery of dsRNA-Cy3 was monitored in ISE6 cells using a Leica TCS SP8 confocal microscope. Briefly, the cells were grown to confluency on 35 mm optical bottom Petri dishes (Fisher Scientific) and incubated with dsRNA-Cy3 polyplexes in a temperature-controlled chamber (Leica, Wetzlar, Germany). Images were captured every 5 min for 2 h and compiled into a motion picture using a Leica TCS SP8 LIGHTNING confocal microscope ran with LASX 2020 software (Leica, Wetzlar, Germany) using a 40× objective.