Vectastain containing 4 6 diamidino 2 phenylindole dapi

Zebrafish expressing DsRed under the control of the cmlc2 promotor^{62 (link)} were treated with atorvastatin or DMSO from tailbud stage on and fixed at 48 hpf with 1% formaldehyde for 50 min at room temperature (RT). After washing with PBS, embryos were incubated for 1 h in blocking solution containing 10% normal goat serum (Vectorlabs, UK), 2 mg/ml bovine serum albumin (BSA) and 0.2% saponin in phosphate-buffered saline (PBS). Primary antibodies diluted in PBS with 0.2% saponin were added to the embryos and incubated overnight at 4 °C. An antibody against DsRed was used to enhance the fluorescence of the DsRed. Atrial myosin was stained with the S46 antibody to distinguish atrial from ventricular cardiomyocytes. Next day, samples were washed several times for 15 min with PBS supplemented with 0.2% saponin and subsequently incubated in secondary antibodies (diluted in blocking solution) for 3 h at RT, followed by several washing steps with PBS. After mounting the embryos with Vectastain containing 4′,6-diamidino-2-phenylindole (DAPI; Vectorlabs) between coverslips, samples were stored at 4 °C until imaging via confocal microscopy.

The cellular uptake of peptide-modified GC/siRNA nanoparticles was assessed next. The HeLa cells were seeded on a cover glass, placed in 12-well non-tissue culture plates (1 × 10⁵ cells/well), and cultured in DMEM containing 10% FBS and 1% PS at 37 °C in a 5% CO₂ atmosphere. Fluorescein isothiocyanate (FITC)-conjugated siRNA was used to prepare nanoparticles, which were added to the plates (100 pmol/mL). The cells were fixed with 4% formaldehyde after 4 h of incubation and were treated with Vectastain^® containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Lysosomal staining was performed using LysoTracker™ Red DND-99 according to the manufacturer’s instruction (50 nM; Invitrogen, Carlsbad, CA, USA). Images were captured using a fluorescence microscope (Nikon Instruments, Melville, NY, USA).

Cells cultured on a cover glass were fixed in 4% paraformaldehyde for 10 min, blocked with 0.5% Triton X-100/PBS for 5 min, and then reacted with the appropriate primary antibodies and Alexa Fluor® 488- and 594-conjugated secondary antibodies. Coverslips were mounted in Vectastain containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Cells were analyzed with fluorescence microscopy (Nikon, Gotenba, Shizuoka, Japan).