2.12 Histological analysis of skin tissue
The RS-NC-injected mouse skin tissue was observed by histological analysis after H&E staining. All animal experiments were performed according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Incheon National University (INU-ANIM-2021-01). First, RS-NC (N/P ratio 5) containing 200 pmol of negative control siRNA were incubated for 30 minutes at RT. 30 µL of RS-NC and DPBS as negative control were injected intradermally to epilated back of each five-week-old BALB/c mouse (Orientbio, Inc., Seongnam, South Korea). After 4 days, the injected skin tissues of the euthanized mice were excised and fixed in 10% (w/v) formalin (Sigma-Aldrich, MO, USA). After 22 hours, the tissue samples were dehydrated, paraffin-embedded by embedding center (Histostar; Thermo Fisher Scientific, MA, USA), and finally sectioned to 4 µm thickness by microtome (HM 355S; Thermo Fisher Scientific, MA, USA), and placed on the slide. After deparaffinization, the tissue samples were stained with hematoxylin and eosin (BioGnost, Zagreb, Croatia). Then, the slides were observed with Leica DM1000 microscope (Leica Microsystems, Hesse, Germany) under 20× magnification.