2.7 Z-stack imaging of RS-NC
The intracellular localization of RS-NC was observed by z-stack imaging. First, 2.0 × 105 MDA-MB-231 cells were seeded to a 35-mm confocal dish (SPL Life Sciences Co., Ltd., Pocheon, South Korea) and incubated overnight in standard conditions. Next, the 200 nM FITC-labeled siRNA was incubated with RS peptide (N/P ratio 5) for 30 minutes at RT. After four-hour incubation with NC, the actin filaments of cells were stained Phalloidin (diluted by PBS-T as 1:200 v/v). The bright-field, fluorescence, and z-stack images were obtained at 900× magnification using a fluorescence confocal microscope (Ti2; Nikon, Tokyo, Japan). The image was analyzed at the single-molecule level using super-resolution radial fluctuation (SRRF). ImageJ software was used to merge the fluorescence images of FITC and Phalloidin.[28]