Las x system

Human glioblastoma cell lines, U87MG and U87MG-shSPARC, were seeded on cover-glass in a 12-well plate (Nalge NUNC International, Rochester, NY, USA; 1 × 10⁵ cells/well), respectively. Cells were incubated with ICG or ICG-HSA complex for 30 min and then washed three times with PBS. Cell fixation by paraformaldehyde (Santa Cruz Biotechnology, Inc.; 300 μL, 10 min/well) and mounting with ProLongTM Gold antifade reagent with DAPI (Invitrogen) and covered the samples with a cover slide. Fluorescence signals were detected using a confocal laser scanning microscope (Leica TCS SP8; Leica, Wetzlar, Hesse, Germany) in the specific range of wavelength (DAPI; 401–480 nm, ICG; 633–800 nm). Fluorescence intensities were analyzed using a LAS X system (Leica Microsystems; Leica).

Confocal imaging was used to analyze the fluorescence signal in the samples. After performing immunofluorescence staining, samples were washed with PBS three times, and the samples were mounted with the ProLong^TM Gold antifade reagent with DAPI (Invitrogen, Grand Island, NY, U.S.A) and covered with a cover slide. The confocal imaging samples were stored at 4 °C and imaged the next day. Fluorescence signals were detected using a confocal laser scanning microscope (Leica TCS SP8m Wetzlar, Hesse, Germany) in the specific range of wavelength (DAPI; 401–480, FITC; 495–519 Cy3; 550–570). Fluorescence intensities were analyzed using the LAS X system (Leica Microsystems, Wetzlar, Germany).