Pe conjugated mouse anti human cd34 antibody

For the expression of the endothelial markers, cells were incubated for 90 min at 4 °C with the antibodies, then washed with PBS and analyzed by the flow cytometer FACSCanto (Becton Dickinson). The antibodies used were as follows: phycoerythrin (PE)-conjugated mouse anti-human CD31 antibody (1:20, BD Biosciences, Milan, Italy); PE-conjugated mouse anti-human CD34 antibody (1:20, clone BIRMA-K3, DakoCytomation, Denmark); PE-conjugated mouse anti-human CD133/1 antibody (1:20, clone AC133, Miltenyi Biotec Inc., Bergisch Gladbach, Germany); PE-conjugated mouse anti-human Tie2 antibody (1:25, R&D Systems, Minneapolis, MN, USA); PE-conjugated mouse anti-human VEGFR2 (KDR) antibody (1:25, R&D Systems) or PE-conjugated mouse IgG₁ isotype control antibody (Miltenyi Biotec Inc.). Data were analyzed with FACS Diva software (Becton Dickinson).

Experiments involving animals were approved according to the Italian law (D. Lgs. 26/2014). After 2 weeks in endothelial culture conditions, GdECs were incubated for 90 min at 4 °C with PE-conjugated mouse anti-human CD34 antibody (1:20, clone BIRMA-K3, DakoCytomation) or PE-conjugated mouse IgG₁ isotype control antibody (Miltenyi Biotec Inc.). Two subpopulations with different CD34 expression levels were isolated by using FACSAria cell sorter (BD Biosciences). Athymic mice (4–6 weeks old; Charles River, Milan, Italy) were implanted subcutaneously with 2 × 10⁵ CD34^high or CD34^−/low GdECs. Briefly, 4-μm sections were obtained from formalin-fixed, paraffin-embedded (FFPE) blocks and after antigen retrieval, were deparaffinized, rehydrated and incubated for 1 h at room temperature with a prediluted mouse anti-human CD34 antibody (1:20, clone BIRMA-K3, DakoCytomation) or a prediluted rabbit anti-GFAP monoclonal antibody (Clone EP672Y; Ventana Inc. Tucson, AZ, USA).