Evos fluorescence imaging system

Fluorescence imaging of live cells (HEK 293 T cells and BJ fibroblasts plated on polyD-lysine coated 35 mm Ibidi μ-dish transduced with different plasmid constructs) was performed 2–3 days after lentiviral transduction using EVOS fluorescence imaging system (Thermo Fisher Scientific) or LSM780 and LSM880 laser-scanning confocal microscope system (Zeiss, Oberkochen, Germany) at 37 °C and 5% CO₂ with a ×60 oil objective. Images were analyzed using Fiji (ImageJ 1.52c), Zen 2.3 (Zeiss, Oberkochen, Germany) and Imaris v9.2 (Zurich, Switzerland) microscopy image analysis software. Images were taken at 3–5 different field locations for each biological replicate.

Immunofluorescence staining was performed as described previously [24 (link)]. Briefly, FFPE tissues of SFT/HPC cases were cut into sections 3 mm thick and deparaffinized using xylene. Heat-induced antigen retrieval was performed in citrate buffer (pH = 6.0) for 15 min, and 3% hydrogen peroxide was applied to inactivate endogenous peroxidase activity. The sections were next incubated for 16 h at 4 °C with primary antibodies targeting CD34 (anti-mouse antibody, 1:50, M7165, DAKO) and CLDN5 (anti-rabbit antibody, 1:200, ab131259, Abcam). Subsequently, sections were co-incubated with goat anti-mouse IgG (1:200, A-11001, Life Technologies, Carlsbad, CA, USA) and goat anti-rabbit IgG (1:200, A1011, Life Technologies) secondary antibodies for 1 h at room temperature. Nuclei were counterstained with 300 nM 4′,6-diamidino-2-phenylindole (DAPI) for 20 min, followed by washing with PBS. The chamber slides were mounted with antifade mounting medium and imaged using the EVOS fluorescence imaging system (Thermo Fisher Scientific, Waltham, MA, USA).