Cd31 antibody

Heart tissue was digested in PBS containing 2 mg/ml Collagenase type II (Worthington), 2.4 mg/ml Dispase II (Sigma‐Aldrich) and 20 µl 1 M CaCl₂ at 37°C for 1 h. After filtration, cardiac ECs were isolated using CD31 antibodies (550274, BD Biosciences) coupled to magnetic beads (Thermo Fisher Scientific). RNA was extracted using the RNeasy mini kit (Qiagen). Libraries were prepared with an input of 10 ng RNA per sample using the SMART‐Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara). Samples were sequenced on a HiSeq 2000 v4 system (Ilumina) with a depth of 50 base pairs in single‐read mode. RNA sequencing data were processed using the DESeq2 package for detection of differentially expressed genes.
For gene set enrichment analysis (GSEA), a list containing all annotated genes detected by RNA‐sequencing with their corresponding log2 fold changes and P‐values was used (17,290 genes). Data were processed using the GSEA software (Broad Institute, Cambridge, MA, USA). Expression levels of genes involved in specific KEGG pathways in individual samples were visualized using heat maps generated with the Morpheus software (Broad Institute, Cambridge, MA, USA).

Penicillin, streptomycin, tetracycline, Dulbecco’s Modified Eagle’s medium, and fetal bovine serum were obtained from Invitrogen (Life Technologies, Carlsbad, CA, USA). The protein assay kit was from Bio-Rad (Hercules, CA, USA). Fibronectin and S100A4 were from Sigma-Aldrich (St. Louis, MO, USA); CD31 antibodies were from BD Biosciences (BD Biosciences, San Jose, CA, USA); antibodies against Jagged 1, and Hes 1, Hey 1, and Hes 5 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Notch 1, N1ICD, RBP-Jκ, and iNOS were from Cell Signaling Technology (Danvers, MA, USA). The fluorescent-700/800 secondary antibodies were obtained from Invitrogen (Carlsbad, CA, USA), an antibody against CD45 was from Millipore (Billerica, MA, USA), and antibodies against F4/80, proliferating cell nuclear antigen, GFP, and rabbit anti-α-SMA were from Abcam (Cambridge, MA, USA). The GFP antibody was purchased from Rockland Immunochemicals (Limerick, PA, USA), and lipopolysaccharide and recombinant IL-4 were purchased from R&D Systems (Minneapolis, MN, USA). BrdU Labeling and Detection Kits were obtained from Roche (Indianapolis, IN, USA).