Anti cd34

Anti-CD34, CD45, CD105, and CD146 antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Anti-CD29 and SSEA-4 antibodies were purchased from BD Bioscience (San Jose, CA, USA). Single-cell suspensions (1–3 × 10⁵) were incubated for 30 min at 4 °C in 100 μl of specific antibody (10 μg/ml). Antibody-labeled cells were washed twice with staining buffer (PBS containing 5% fetal calf serum (FCS) and 0.01% sodium azide) and incubated in 20 μg/ml of FITC-conjugated goat anti-mouse IgG for 20 min at 4 °C. Cells were then washed two times with staining buffer and either analyzed immediately or fixed with 1% paraformaldehyde and analyzed within 96 h using a Becton Dickinson FACStar^plus (Franklin Lakes, NJ, USA) flow cytometer with 10,000 events collected per sample. The percentage of positive cells was determined by fluorescence-activated cell sorting (FACS). All assays were run in parallel using cells stained with isotype IgG as a negative control.

Primary antibodies included: anti-α-actin (Abcam, ab5694; IHC and IF, 1:100; WB, 1:1000); Dako actin (Smooth Muscle) Clone 1A4; anti-cleaved Caspase-3 (Cell Signaling #9661; IHC, 1:50; WB, 1:1000); anti-CD31 (Abcam, ab28364; IHC and IF, 1:50); anti-CD34 (R&D, AF4117; IF, 1:100; WB, 1:1000); anti-CD68 (ED1; Abcam, ab31630; IHC, 1:200; WB, 1:1000); anti-Eph-B4 (Santa Cruz, sc-5536; IF, 1:50); anti-Ephrin-B2 (Novus, NBP1-48610; IHC, 1:50); anti-GAPDH (Cell Signaling, 14C10; WB, 1:2000); anti-IL-10 (Abcam, ab9969; IF, 1:100); anti-Ki67 (Abcam, ab15580; IHC and IF, 1:100; WB, 1:1000); anti-phospho-mTOR (Cell Signaling, #2971; IHC, 1,50; WB,1:1000); anti-mTOR (Cell Signaling, #2972; WB, 1:1000); anti-transglutaminase 2 (TGM2; #37557; IF, 1:100); anti-VEGFR2 (ABCAM, ab2349; IF, 1:100; WB,1:1000); Secondary antibodies used for IF were: donkey anti-goat Alexa-Fluor-488, donkey anti-rabbit Alexa-Fluor-488, donkey anti-rabbit Alexa-Fluor-568, donkey anti-mouse Alexa-Fluor-568 and chicken anti-mouse Alexa-Fluor-488 conjugated antibodies from Invitrogen (1:1000). For IHC, sections were incubated with EnVision reagents for 1 h at room temperature and treated with Dako Liquid DAB + Substrate Chromogen System (Dako). Finally, the sections were counterstained with Mayer’s hematoxylin.