4 6 diamidino 2 phenylindole solution

Immunofluorescence was performed on 4-um-thick paraffin sections. After deparaffinization and rehydration, antigen retrieval with target retrieval solution (Dako, Carpinteria, CA, USA) was performed. The sections were blocked and incubated with diluted primary antibody (Dako) at 4 °C overnight. Mouse anti-TNNT2 (Abcam; 1:200, Cambridge, UK) antibody was used to stain myocardium. Next, the samples were incubated with anti-mouse immunoglobulin G Alexa Fluor 488 (Invitrogen; 1:500) secondary antibody for 60 min at room temperature and then stained with 4′,6-diamidino-2-phenylindole solution (VECTOR, Torrance, CA, USA) for nuclear staining. Images of the heart sections were visualized using an LSM 800 laser scanning microscope with Airyscan processing (Zeiss, Oberkochen, Germany).

To verify the target-specific binding potential of anti-EGFR iEDNs, 100 μg of EDNs or iEDNs was used to treat MDA-MB-231 (EGFR+) and MDA-MB-453 (EGFR−) cells in 6-well plates, which were further cultured for 24 h. The treated cells were washed twice with L-15, harvested with trypsin-EDTA, and centrifuged at 200× g for 3 min. The cell pellets were resuspended with the culture media in 5 mL polystyrene round-bottom tubes (Corning, New York, NY, USA). Alexa Fluor 488 antibody (Invitrogen, Eugene, OR, USA) was used to label the anti-EGFR antibodies for 10 min at 4 °C with continuous agitation. The binding of EDNs and iEDNs to the cells was analyzed using a FACSCalibur flow cytometer (Becton Dickinson).
To verify the cellular uptake of iEDNs-DOX, the same cancer cells were treated with 100 μg of iEDNs-DOX and incubated for 1, 4, 8, 12, or 24 h at 37 °C in a serum-free medium. After treatment, all the cells were washed twice with PBS (pH 7.4, ice-cold) and fixed with 2% paraformaldehyde at 4 °C for 10 min in the dark. After washing thrice with PBS, the cells were stained with 4’,6-diamidino-2-phenylindole solution (Vector Lab, Burlingame, CA, USA) for 10 min in the dark and mounted on slides. The slides were observed using a confocal laser scanning microscope (LSM 510; Zeiss).