Evos amg cell imaging system

Imaging for Masson’s trichrome assay, biotinylated isolectin B4 staining, and for PDGFRα⁺ and NG2⁺ cells in skeletal muscle was acquired with a Leica CTR 5500 microscope and DFC425 camera for bright-field images, a DFC340FX camera for immunofluorescence pictures or with an EVOS AMG cell imaging system (Thermo Fisher Scientific). For the aortic ring experiments and fluorescence imaging of differentiated and undifferentiated GFP⁺PDGFRα⁺ cells, imaging was performed with an EVOS AMG cell imaging system (Thermo Fisher Scientific).
Quantification of microvessel density and vessel breakdown in MCM⁺/iDTR^+/− mice was performed by counting in 4 defined microscope fields in 3 different samples per condition. Measurements were performed on an identically sized area for all samples (0.1mm²).
Maximal fibrotic area was measured with ImageJ. Slides for each condition after HLI (n = 3) were analyzed under the microscope to find the minimal and maximal fibrotic area covering the entire extension of injury. The maximal fibrotic area for the adoptive transfer and the ablation studies was quantified using values of maximal threshold depending on the level of injured tissue in the animals analyzed.

An aortic ring assay was performed as described (Baker et al., 2011 (link)). Briefly, freshly isolated aortas from PDGFRαH2B-eGfp mice were cut in cross-section into 1μm ring segments and distributed on 50 μL of low growth factor matrigel (Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, Corning). Aortic rings and matrigel were left at 37°C for 30 min to allow the matrigel to solidify prior adding EBM media supplemented with VEGF-AA (30ng/ml, Peprotech) or PDGF-AB (30ng/ml, R&D). Sprouting of cells was observed for 5d and recorded on an EVOS AMG cell imaging system (Thermo Fisher Scientific). After 5d, the rings were fixed with 4% PFA, permeabilized with PBS/Triton 0.25% and stained with NG2 as described above.