Apg500

Immunohistochemistry was performed as described above, with some changes. Cells were fixed in freshly prepared 2% paraformaldehyde in Hank’s balanced salt solution (HBSS) for 15 min at room temperature; then, nonspecific binding was blocked with HBSS containing 10% normal donkey serum (017-000-121, Jackson Immunology, Cambridgeshire, UK) and 0.1% Triton X-100 for 1 h at room temperature. Next, cells were incubated with anti-VEGFR2 (AF357, 1:100) or anti-NRP2 (AF2215, 1:100), both from R&D Systems, anti-VE-cadherin (361900, Invitrogen, 1:100), and anti-CD34 (Sanquin, 1:100) diluted in primary antibody diluent (APG500, Scytek, Logan, UT, USA) for 1 h at room temperature. The secondary donkey anti-rabbit Alexa647 Plus, donkey anti-mouse Alexa488 Plus (both from Invitrogen, 1:1000) or donkey anti-goat Cy3 conjugated antibody (1:200, Jackson Immunology) were also diluted in antibody diluent and incubated for 1 h at room temperature. Cells were mounted in Vectashield containing DAPI. Images were recorded using a confocal laser scanning microscope (SP8; Leica), and fluorescence intensity was quantified using ImageJ software with subtraction of the background signal of five images per condition.

Four‐micrometre‐thick tissue sections were cut from selected paraffin‐embedded blocks (Superfrost Microscopic Slides; ThermoFisher Scientific, Bleiswijk, The Netherlands). Slides were deparaffinised, and rehydrated with xylene and ethanol. Endogenous peroxidase was blocked with 0.3% H₂O₂ in phosphate‐buffered saline, and heat‐induced antigen retrieval was accomplished by 15 min of incubation in Tris‐EDTA buffer (pH 9; Klinipath, Duiven, The Netherlands). Mouse monoclonal high molecular weight cytokeratin (clone 34BE12; 1:200; Dako, Heverlee, Belgium) diluted in normal antibody diluent (APG‐500; ScyTek Laboratories, West Logan, WV, USA) was incubated for 2 h at room temperature. Antibody visualisation was performed with the Envision kit (Dako) and slide counterstaining with haematoxylin. When basal cell staining was absent, the cribriform structure was classified as invasive carcinoma; if sporadic, scattered or continuous basal cells were identified, the growth pattern was classified as intraductal carcinoma.