2.8. Cellular internalization observed using confocal microscopy
The cellular internalization of siRNA/peptide complex was investigated using confocal images. HeLa cells of 2.0×104 were incubated in a 35 mm confocal dish (SPL Life Sciences Co., Ltd., Pocheon, South Korea) for 24 hours. After 30-minute incubation of final 50 nM Cy3-labeled siRNA and fluorescein (FITC)-labeled S-R15 (20:1 N/P ratio), the complex was applied to the cells for 4 hours. The nucleus and actin were stained using 5 μg/mL Hoechst 33342 and 100 nM SiR-actin kit, respectively. The intracellular localization and co-localization of siRNA and S-R15 were confirmed using fluorescence and confocal microscope (Ti2; Nikon, Tokyo, Japan). Each of them was analyzed at the single-molecule level using super-resolution radial fluctuation (SRRF). Bright-field and fluorescence images were acquired at 900× magnification. Fluorescence images of Cy3, FITC, and SiR-actin were merged using Image J software (Schneider et al., 2012).