Immunohistochemical analysis of VEGF Receptor 1/2 in formalin-fixed paraffin-embedded human breast carcinoma tissue section. Samples were incubated in VEGF Receptor 1/2 polyclonal antibody (Product # PA1-16612) using a dilution of 1:500. Bond Rx autostainer (Leica Biosystems). The assay involved 20 minutes of heat induced antigen retrieval (HIER) with 10mM sodium citrate buffer (pH 6.0) and endogenous peroxidase quenching using peroxide block. The sections were incubated with primary antibody for 30 minutes. Bond Polymer Refine Detection (Leica Biosystems) and DAB were used for signal detection which followed counterstaining with hematoxylin. Whole slide scanning and capturing of representative images (20X) were performed using Aperio AT2 (Leica Biosystems). This VEGFR1/Flt-1 antibody generated an expected membrane cytoplasmic staining of VEGFR1 protein in the cancer cells. The tumor stroma/stromal cells did not show VEGFR1/Flt-1immunopositivity. Staining was performed by Histowiz.