Zen blue pro

Native and decellularised zinc fixed and paraffin embedded tissue sections (from n = 6 human aortas) were stained with monoclonal antibodies against collagen I (Millipore; IgG isotype, polyclonal, 1:500 dilution), collagen III (Millipore; IgG1 isotype, clone IE7-D7, 1:100 dilution), collagen IV (Dako; IgG1 isotype, clone PHM-12, 1:200 dilution), von Willebrand factor (Dako; IgG isotype, polyclonal, 1:400 dilution), fibronectin (Vector; IgG1 isotype, clone 568, 1:150 dilution) and laminin (Sigma; IgG1 isotype, clone LAM-89, 1:1000 dilution). Immunolabeling was carried out using an Ultravision detection system (Thermo Scientific). Antigen retrieval was employed using proteinase K (Dako; room temperature for 20 min—collagen III, von Willebrand factor, fibronectin and laminin), proteinase K followed by trypsin digestion (0.05% w/v trypsin 0.52 mM EDTA.4Na in HBSS with phenol red (Sigma) for 30 s) (collagen IV) and Vector antigen unmasking solution citric based (collagen I). Hydrogen peroxide (Sigma) was used to block endogenous peroxidase activity. Isotype control antibodies (IgG and IgG1; Dako; used at the same concentrations as the test antibody) and omission of the primary antibody served as negative controls. Sections were viewed using Kohler and all images were captured digitally using Zen Blue Pro (Zeiss).

Native and decellularised tissue samples from each of six different human aortas were fixed in zinc fixative solution (0.5% w/v zinc chloride (VWR Chemicals), 0.5% w/v zinc acetate (VWR Chemicals), in 0.1 M Tris buffer containing 0.05% w/v calcium acetate (Sigma) at pH 7.4) for 48 h, dehydrated, and embedded in paraffin wax using a Leica Tissue Processor (TP1020). Serial sections (6–8 µm) were stained with haematoxylin and eosin (H&E), picrosirius red/Millers elastin (to visualize collagen and elastin) and alcian blue (glycosaminoglycans, GAGs) using standard histological methods. Cell nuclei were visualised using DAPI dye (Sigma Aldrich); briefly, sections of tissue were rehydrated in a graded alcohol series before they were incubated in the dark in 0.1 µg mL⁻¹ DAPI dye in dye buffer (10 mM Tris, 1 mM EDTA, 1 mM sodium chloride in water) for 10 min. The sections were viewed using an upright Zeiss Imager M2 microscope under normal Kohler illumination or under fluorescent illumination using a DAPI filter (λ_ex = 340–380 nm/λ_em = 435–485 nm) and images were captured digitally using Zen Blue Pro (Zeiss).