Vegfr2

Paraffin-embedded sections from re-endothelialized scaffolds and transplanted scaffolds were rehydrated, and antigen retrieval was performed. Briefly, the slides were boiled in 10 mM citrate buffer with 0.05% Tween-20 at pH 6.0 for 20 minutes and then blocked in 3% BSA in PBS for one hour. Then, we incubated the slides with 10 ug/mL of the appropriate primary antibody in PBS overnight at 4°C. We used antibodies to proliferating cell nuclear antigen (PCNA) (rabbit polyclonal, Santa Cruz), CD31 (rabbit polyclonal, Santa Cruz), endothelial nitric oxide synthase (eNOS), calretinin, vimentin (rabbit polyclonal, Abcam), vascular endothelial growth factor receptor 2 (VEGFR2; mouse monoclonal, BD Bioscience), CD34, CD45 (mouse monoclonal, Santa Cruz), α-smooth muscle actin (mouse monoclonal, Sigma), and von Willebrand factor (vWF; rabbit polyclonal, Abcam; goat polyclonal, Santa Cruz). The slides were washed between steps with three changes of PBS containing 0.05% Tween-20 and incubated for one hour with the appropriate secondary antibody conjugated with either FITC or Texas Red (Jackson Immunoresearch) at a 1∶250 dilution. We mounted the slides with DAPI-containing mounting medium and examined them on a Nikon Eclipse TE200 fluorescent microscope.

The enzyme-linked immunospot (ELISPOT) assay was performed to assess IFN-γ secretion. Briefly, splenocytes (2 × 10⁵/well) were incubated in polyvinylidene difluoride (PVDF)–bottom 96-well filtration plates (Dakewe Biotech Company Limited) at 37 °C with 5% CO₂ in the presence or absence of VEGFR-2 and TERT proteins (BD Pharmingen) (0.5 mg/ml VEGFR-2 with 0.5 mg/ml TERT or 0.25 mg/ml VEGFR-2 with 0.25 mg/ml TERT) for 4 h. Once IFN-γ was produced, wells were read using an ELISPOT reader (Cellular Technology Ltd.). The numbers of spot-forming cells (SFC) per 10⁶ cells was calculated.

One ×105 CFU-F-derived cells were incubated with 5 µL of FITC or PE-anti-human-CD34, CD45, CD73, CD90, CD31, CD105 and class I-HLA monoclonal antibodies and appropriate isotype (all from Beckman Coulter, Milan, Italy). Cell acquisition was performed by Navios flow cytometer (Beckman Coulter) and analysis was performed by the Kaluza software, 2.1 version (Beckman Coulter). Similarly, 1 × 105 ECFC-derived cells were incubated with anti-human CD45, CD34, CD146, CD31, CD144, VEGFR2 and CD105 (all from BD, Pharmingen, San Diego, CA, USA). The percentage of positive cells was calculated, subtracting the value of the appropriate isotype controls.

Retinas were dissected and stained as previously described [66 ]. The following antibodies were used at a 1:100 concentration: αSMA (Sigma C6198), cleaved CASPASE-3 (Cell signaling 9661), COLLAGEN IV (Millipore AB756P), ENDOMUCIN (Santa Cruz 6415), ERG (Abcam 92513), GFP (Aves GFP-1020), KI67 (Cell Signaling 9449), NG2 (Millipore 5320), PECAM/CD31 (BD 553370), VEGFR2 (BD 555307). Additionally, the following immunofluorescent stains were performed according to the manufacturer’s instructions: Dapi (Life Technologies R37606), Isolectin-488 (Invitrogen 21411), Isolectin-594 (Invitrogen 21413), Isolectin-647 (Invitrogen 32450). Confocal images were taken at the same exposure settings for both mutant and control retinas, so fluorescent intensity could be compared.

Cultured EPCs were identified and characterized by flow cytometry using hematopoietic stem cell markers CD34, CXCR4, c-Kit, VEGFR2, and VE-Cadherin were purchased from (BD Pharmingen, Franklin Lake, NJ, USA). The flow cytometry analysis was carried out by fluorescence activated cell sorting (FACS; BD FACS canto 2, San Joes, CA, USA). The percentage of stained cells was indicated by the red line peaks.