Proteome profiler mouse angiogenesis array kit

To analyze the expression of angiogenesis-related proteins in uterine tissue samples, a proteome profiler mouse angiogenesis array kit was used according to the manufacturer´s instruction (R&D Systems, Wiesbaden, Germany). In brief, pooled uterine tissue samples were stored in lysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% Triton-X 100, 0.02% NaN₃, 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 1:75 v/v protease inhibitor cocktail and 1:100 v/v phosphatase inhibitor cocktail (all from Sigma-Aldrich; Taufkirchen, Germany)) and homogenized. The tissue lysate was then incubated for 30 min on ice and afterwards centrifuged at 4 °C for 5 min at 16,000×g. The supernatants were used for whole protein extracts. A total of 250 µg protein per group was used for the array. The samples were mixed with the biotinylated detection antibody cocktail and incubated for 1 h at room temperature. Subsequently, the mixture was exposed over night at 4 °C to the capture antibodies-spotted array membrane. The visualization of the labeled specific target proteins was achieved with streptavidin–horseradish peroxidase and chemiluminescent detection reagents using an Intas ECL Chemocam Imager (Intas Science Imaging Instruments GmbH, Göttingen, Germany).

Gelatin (porcine skin, type‐A powder), paraformaldehyde, and streptozotocin (STZ) were purchased from Sigma‐Aldrich (USA). Collagenase type IV, Collagenase type II, and DNase I were purchased from Sangon Biotech (China). DAPI, Red Blood Cell Lysis Solution, and Citrate Antigen Retrieval solution were provided by Beyotime (China). Mouse CD45 MicroBead Kit and Human CD14 MicroBead Kit were purchased from Miltenyi Biotec (Germany). Proteome Profiler Mouse Cytokine Array Kit and Proteome Profiler Mouse Angiogenesis Array Kit were purchased from R&D Systems (USA). RayBio^® L‐Series Mouse Antibody Array 308 Membrane Kit was provided by RayBiotech (USA). Corning^® Matrigel^® Growth Factor Reduced (GFR) Basement Membrane Matrix was provided by Corning (USA).

Culture media from EOMA cells and MBMECs, alone or co-cultured with platelets for 24 hours, were collected and centrifuged. The supernatants were used to detect the levels of released angiogenic factors using the Proteome Profiler Mouse Angiogenesis Array Kit (ARY015, R&D Systems) according to the manufacturer’s instructions.