Immunofluorescence Staining of Flt1 (VEGFR1)

Immunofluorescence was performed using a heat-induced epitope retrieval (HIER) protocol with slight modification^{49 (link)}. After deparaffinization, slides were placed in a plastic container filled with citrate buffer, pH 6.0 at 60 °C for 20 min. And then, slides were allowed to cool for 20 min at room temperature and were then rinsed with phosphate-buffered saline with Tween 20 (PBST, Duchefa Biochemie, Hofmanweg, Netherlands). Slides were incubated in blocking buffer (BLOXALL® Endogenous Peroxidase and Alkaline Phosphatase Blocking Solution, Vector Laboratories, Inc., California, USA) for 1 h at room temperature to remove non-specific binding. Next, they were incubated for 24 h with Flt1 (VEGFR1) antibody (Santacruz biotechnology, Texas, USA) in blocking buffer at 4 °C. Next day, the slides were washed and incubated for 1 h at room temperature with anti-mouse secondary antibodies conjugated with Alexa Flour 647 (Abcam, Cambridge, United Kingdom). The slides were counterstained with DAPI for 10 min and mounted with fluorescence mounting medium (Agilent, California, USA).