Wga alexa fluor 488 conjugate membrane staining dye

MDA-MB-231 cells were seeded on micro cover glass (VWR, Pennsylvania, USA) in 6 cm petri dishes and cultured in 10% FBS DMEM medium at 37͘ °C. Cells were separated into three exposure duration groups, 0, 3, and 6 h, to evaluate reagent retention inside the cells at each time point. When the cells reached sub-confluency in each petri dish, VP or HS201 (1.0 μM) was added, co-incubated for 30 min, removed, and washed once with PBS. For the 0-h group, cells were fixed with 10% neutral buffered formalin for 15 min at 37 °C immediately after nIR staining. As for the 3 and 6-h group, culture media were exchanged every 1, 3, or 6 times, respectively, before the cells were fixed. After the formalin was removed, the cells adherent to the micro-glass were stained with wheat germ agglutinin (WGA) Alexa Fluor 488 conjugate membrane staining dye (Invitrogen, Massachusetts, USA) and DAPI (BioLegend, California, USA) for 10 min at room temperature. After being washed, micro cover glasses were mounted on the glass slides and observed using a ZEISS LSM880 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Imaris for Cell Biologists—CL software (Bitplane, Zurich, Switzerland) was used to generate 3D images.

VP or HS201 (500 nmol/mouse) was injected into tumor-bearing mice via tail vein as described above. Mice were sacrificed at designated time points (1, 3, 6, or 12 h) after compound injection and their tumors were harvested. A mouse without any injection was also sacrificed as a control. Tumors in some mice were irradiated with laser light (690 nm wavelength, 120 J/cm²/4 min) using ML7710 laser system (Modulight) at 6-h time point after the PS injection, and were collected at 12 h time point. Tumors were fixed overnight in 10% neutral buffered formalin before sectioning into 50 μm thick sections using Compresstome^TM Microtome. Samples were stained using WGA Alexa Fluor 488 conjugate membrane staining dye (Invitrogen) and DAPI. A ZEISS LSM880 confocal microscope (Zeiss systems, Germany) with a 40×/1.2NA oil objective was used to collect tiled images of DAPI (e.g. 405 nm/em, 410–450 nm), WGA (e.g., 488 nm/em, 490–530 nm), and VP/HS201 (e.g., 633 nm/em, 635–760 nm). Mean fluorescence intensities of the PS for the whole tumor tissue areas were calculated using FIJI software (version 2.0.0-rc-69/1.52p).