Anti rabbit horseradish peroxidase labeled polymer

Hematoxylin and eosin staining and immunostaining were used to evaluate the histology of the tumors formed by the tested cells. First, xylene was used to remove the formalin, followed by alcohol stratification and rehydration. The antigen unmasking solution (Dako, Agilent, Santa Clara, CA, USA) and 3% hydrogen peroxide in distilled water were used to block the intrinsic peroxidase activity. Immunostaining was performed with antibodies against PAX8 (1:500, Dako) and WT-1 (1:500, Dako), CK7 (1:500, Dako) according to the manufacturer’s instructions (Vector). The specimens were then placed in a refrigerator at 4 °C overnight and treated with anti-rabbit horseradish peroxidase-labeled polymer (Dako) at room temperature for 30 min. All immunostained slides were stained with hematoxylin. WT-1 positive cells were calculated by the positive staining cell numbers among 50 cells counted in each of the three fields.

To determine the histological localization of Tie2 protein in distinct microvascular compartments, immunohistochemical staining of Tie2 was performed as previously described [18 (link)]. Briefly, 4 μm acetone-fixed cryosections were incubated with primary monoclonal rat-anti-Tie2 antibody (Tek4, IgG1 isotype; eBioscience, Thermo Fisher Scientific, San Diego, CA, USA), followed by rabbit-anti-rat IgG1 antibody (Vector Laboratories, Burlingame, CA, USA), and anti-rabbit, horseradish peroxidase-labeled polymer (Dako, Heverlee, Belgium). Peroxidase activity was detected with 3-Amino-9-ethylcarbazole (Sigma-Aldrich). Sections were counterstained with Mayer’s hematoxylin (Merck, Darmstadt, Germany). Isotype control staining with rat IgG1 (Antigenix America, New York, USA) was consistently negative. All sections of a given organ (n = 15 mice) were stained in one experiment to avoid inter-experiment staining variability.

To determine the location of Tie2, E-selectin, and VCAM-1 proteins in distinct microvascular compartments in mouse kidneys and lungs, immunohistochemical staining was performed as previously described [22 (link)]. Incubation with primary monoclonal rat-anti-Tie2 antibody (Tek4, IgG1 isotype; eBioscience, Thermo Fisher Scientific), rat-anti-E-selectin antibody hybridoma supernatant (clone MES-1, IgG2a isotype, a kind gift of Dr Derek Brown, UCB Celltech, Belgium), or rat-anti-VCAM-1 antibody (clone M/K-2, IgG1 isotype, Merck Millipore, Amsterdam, The Netherlands) was followed by mouse absorbed rabbit-anti-rat antibody (Vector Laboratories, Burlingame, CA, USA), and anti-rabbit, horseradish peroxidase-labeled polymer (Dako, Heverlee, Belgium). 3-Amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO, USA) was used for detection, followed by counterstaining with Mayer’s hematoxylin (Merck, Darmstadt, Germany). Isotype control staining with rat IgG1/IgG2a (Antigenix America, New York, NY, USA) was consistently negative (data not shown). Sections of kidneys and lungs (n = 30 mice) were stained in one experiment to avoid inter-experiment staining variability.