Cholera toxin b subunit alexa fluor 647

4T1 murine breast tumor cells were seeded in 6-well plates with sterile coverslips (2.5 × 10⁵ cells/well) at 24 h before treatment. Cells were exposed to 3μM of DOX, SpHL-DOX, and SpHL-DOX-Fol for 24 h. After, the treatments were withdrawn and wells were washed with PBS buffer. Cells were fixed with 3.7% (v/v) formaldehyde and permeabilized with 0.1% (v/v) Triton X-100 solution [26 (link)]. Cell membranes and nuclei were labeled with fluorescent probes Cholera toxin B subunit-Alexa Fluor® 647 (Invitrogen - Carlsbad, USA) and Hoechst 33258 (Thermo Fisher Scientific - Waltham, USA), respectively. The coverslips were washed with PBS and slides were assembled using Prolong Gold Antifade Reagent (Thermo Fisher Scientific - Waltham, USA). Cells were analyzed in “Centro de Aquisição e Processamento de Imagens da UFMG (CAPI/UFMG)” using the LSM 880 microscope with Airyscan detector (ZEISS - Oberkochen, Germany). For image acquisition, it was used 40x objective lens. The lasers used were: Diode 405 nm (excitation of Hoechst 33258), Argonium 488 nm (excitation of DOX) and HeNe 633 nm (excitation of Alexa Fluor®647). The images were processed using the ZEN Blue Edition software version 2.3 lite (ZEISS - Oberkochen, Germany).