Axioimager a1

We analyzed the cell internalization using flow cytometry (Attune Nxt flow cytometry, ThermoFisher, USA) and fluorescence microscopy (AxioImager. A1, Zeiss, Germany). To facilitate the observation, PLGA-Rhodamine was mixed with PLGA at 1:20 w/w ratio during the synthesis process to obtain the fluorescence-labeled FuNPs. After the incubation of FuNPs with 4T1 and MDA-MB-231 cells for 24 h and 48 h in a 6-well plate, the cells (1 × 10⁵) were collected and measured using flow cytometry.
For fluorescence microscopy, 4T1 cells were seeded and allowed to grow on Millicell EZ SLID (Merck, Germany) overnight. After that, we incubated the cells with FuNPs for 24 h and washed the cells twice with PBS. To generate cellular permeability, triton X-100 (0.3% in PBS buffer) was added for 30 min. The cells were then blocked using FBS buffer (30 min), immobilized using anti-F-actin at 4 °C overnight, and further stained using a secondary antibody (1:250) for 1 h. After an 1 h incubation, the cells were washed twice with PBS and stained with DAPI (1 µg mL⁻¹) for 5 min. The cells were then observed using a fluorescent microscope.

Prior to detection of EphB4, CD31, or LYVE-1 by immunohistochemistry, tumor cryosections were fixed with acetone for 10 min at −20°C. After blocking with 3% v/v H₂O₂ for 10 min (only for EphB4) and with 2% v/v bovine serum albumin and 0.3% w/v skimmed milk for at least 1 h, tumor sections were incubated with primary antibodies (goat anti-human EphB4 IgG, R&D, AF3038, 0.2 µg/mL; rat anti-mouse CD31 IgG, BD, 550274, 0.08 µg/mL; rabbit anti mouse LYVE 1 IgG, ReliaTech Hamburg, Germany, 705 065 003; 1:200, 1 h, room temperature), ExtrAvidin^®-Peroxidase (Sigma Aldrich; 1:50, 30 min, room temperature), and AEC Substrate Kit (BD). Primary antibodies for CD31 and LYVE 1 were visualized by Alexa Fluor 488-conjugated goat anti-rat IgG (Thermo Fisher Scientific, Langenselbold, Germany, A11006; 10 µg/mL) and Alexa 546-conjugated donkey anti rabbit IgG (Thermo Fisher Scientific, A10040, 10 µg/mL, LYVE-1), respectively, for 1 h at room temperature and subsequent fluorescence microscopy using the microscope AxioImager.A1 (Zeiss, Jena, Germany) and software AxioVision (Zeiss). Distribution of fluorescence dye Hoechst 33342 was analyzed without further processing of tumor cryosections by fluorescence microscopy. Subsequent to fluorescence microscopy, tumor cryosections were stained with Hematoxylin and Eosin (H&E).