Anti cd31 clone mec13

Cells from adult mice were isolated as previously described [13 (link)]. Briefly, mice were euthanized, and organs were excised, minced, and digested with dispase II (Thermo Fisher Scientific), collagenase (Wako, Osaka, Japan), and type II collagenase (Worthington Biochemical Corp., NJ, USA) with continuous shaking at 37 °C. The digested tissue was filtered (40 μm filters) to obtain single-cell suspensions. Erythrocytes were lysed with ammonium-chloride-potassium buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2-EDTA). Bone marrow cells were collected from the tibiae and femurs. Cell surface antigen staining was performed with anti-CD31 (clone MEC13.3, BD Biosciences, CA, USA) and anti-CD45 (Clone 30-F11, BD Biosciences) antibodies. Hoechst staining was performed as described previously [17 (link)]. Briefly, cell suspensions were incubated with Hoechst 33342 (5 mg/ml) (Sigma-Aldrich Japan) at 37 °C for 90 min in DMEM (Sigma-Aldrich Japan) supplemented with 2% FCS and 1 mM HEPES at a concentration of 1 × 10⁶ nucleated cells/ml in the presence or absence of verapamil (50 mM, Sigma-Aldrich Japan). Propidium iodide (PI, 2 mg/ml; Sigma-Aldrich Japan) was added before FACS analysis to exclude dead cells. Stained cells were analyzed and sorted by a SOAP FACSAria (BD Bioscience), and data were analyzed using FlowJo Software (Treestar Software, San Carlos, CA, USA).

Formalin-fixed paraffin embedded tissue sections were stained with antibodies against CD3 (rabbit polyclonal, catalogue #ab5690; Abcam), mouse α-smooth muscle actin (clone 1A4; Sigma-Aldrich), anti-Fibronectin (FN) (clone AB-10; Neomarkers), anti-CD31 (clone MEC 13.3) and anti-CD45 (clone 30 F11), both from BD Biosciences according to the manufacturer’s protocols.
Corresponding secondary horse-radish peroxidase-conjugated antibodies (DAKO, Glostrup, Denmark) were used, followed by incubation with chromogenic substrate 3,3′-diaminobenzidine (DAKO).
For double staining, secondary antibodies coupled to Alexa Fluor 488 or 568 (1:1500) were purchased from Molecular Probes. Sections were examined by means of confocal microscopy on a LSM 510 (Carl Zeiss Inc).
The blood vessel density was determined by quantifying CD31+ capillaries in 3-4 fields from two different sections of the tumor (magnification, ×200).
Quantification of CD3+ T-cells in tumors from 12-week-old PyMT mice and in pre-metastatic lungs was performed as described [13 (link)].

For staining of SKL cells (Sca-1+ c-Kit+ Lin–), VSELs (Sca-1+ Lin– CD45–), MSCs (Lin– CD45– CD31– CD90+), and EPCs (Lin– CD45– CD31+) the following monoclonal antibodies were used: FITC–anti-CD117 (also known as c-Kit, clone 2B8; BioLegend, San Diego, CA, USA) and PE–Cy5–antimouse Ly-6 A/E (also known as Sca-1, clone D7; eBioscience, San Diego, CA, USA). All anti-mouse lineage marker antibodies, including anti-CD45R (also known as B220, clone RA3-6B2), anti-Ter-119 (clone TER-119), anti-CD11b (clone M1/70), anti-T cell receptor β (clone H57–597), anti-Gr-1 (clone RB6-8C5), anti-TCRγδ (clone GL3), and anti-CD45 (clone 30-F11), conjugated with PE; anti-CD31 (clone MEC 13.3), conjugated with APC; and anti-CD90.2 (clone 30-H12), conjugated with BV510, were purchased from BD Biosciences. Staining was performed in RPMI-1640 medium containing 2% FBS. All monoclonal antibodies were added at saturating concentrations, and the cells were incubated for 30 min on ice, washed twice, and analyzed with an LSR II flow cytometer (BD Biosciences) [15 (link), 17 (link)].