focused ion beam (fib)/sem imaging and scanning transmission electron microscopy (stem) specimen preparation  (Carl Zeiss)

Journal: STAR Protocols

Article Title: Protocol to decellularize porcine right ventricular outflow tracts using a 3D printed flow chamber

doi: 10.1016/j.xpro.2024.102899

Figure Lengend Snippet: Characterization steps to validate the decellularization process Histological assessment (hematoxylin and eosin (H&E), elastin Van Gieson (EVG), and 4′,6-diamidino-2-phenylindole (DAPI)) was carried out on all fresh native samples (fresh RVOT), freeze-thawed native samples (F-T RVOT), fresh decellularized samples (fresh dRVOT), and freeze-thawed decellularized samples (F-T dRVOT) as quality control. Following this, a subset was taken forward for further evaluation by immunohistochemistry, scanning electron microscopy, and DNA quantification. Tensile testing was carried out on F-T RVOTs and F-T dRVOTs.

Article Snippet: Scanning electron microscopy specimen stubs , Agar Scientific , Cat# AGG301.

Techniques: Control, Immunohistochemistry, Electron Microscopy

Journal: STAR Protocols

Article Title: Protocol to decellularize porcine right ventricular outflow tracts using a 3D printed flow chamber

doi: 10.1016/j.xpro.2024.102899

Figure Lengend Snippet: Representative results from scanning electron microscopy of fresh RVOT versus freeze-thawed (F-T) RVOT versus F-T and decellularized (d)RVOT surface topologies from the pulmonary artery (PA), right ventricle (RV), and pulmonary valve (PV) leaflet surfaces at increasing magnifications The native internal pulmonary artery surface is covered with a uniform endothelial cell layer resembling cobblestones, which becomes pitted in appearance upon freezing-thawing, indicating the start of mechanical cell destruction. The decellularized pulmonary artery internal luminal surface lacks this typical cobblestone morphology, with the cells having been removed and the underlying fibrous matrix uncovered. The external pulmonary artery surface is covered by an adventitial layer of collagen fibers, which are retained after the freeze-thawing process but removed by decellularization. Importantly, the underlying cells beneath the adventitia are also disrupted and are shown to be lifting off the underlying extracellular matrix. The native right ventricle surface has a pronounced confluent cell layer, which becomes pitted upon freeze-thawing the tissue. This monolayer is disrupted upon decellularization, with the underlying matrix becoming exposed and the cells removed. The pulmonary valve leaflet surface similarly shows a uniform cobblestone appearance in its native state, resembling that of the pulmonary artery, which is damaged upon freeze-thawing and completely removed following decellularization. Fresh dRVOT images are not shown for clarity. Fresh RVOT, n = 3; F-T RVOT, n = 3; F-T dRVOT, n = 3. Scale bars = 100 μm, 20 μm, or 10 μm, as indicated.

Article Snippet: Scanning electron microscopy specimen stubs , Agar Scientific , Cat# AGG301.

Techniques: Electron Microscopy

Journal: STAR Protocols

Article Title: Protocol to decellularize porcine right ventricular outflow tracts using a 3D printed flow chamber

doi: 10.1016/j.xpro.2024.102899

Figure Lengend Snippet:

Article Snippet: Scanning electron microscopy specimen stubs , Agar Scientific , Cat# AGG301.

Techniques: Recombinant, Staining, Saline, Protease Inhibitor, Picogreen Assay, Software, Membrane, Adhesive, Blocking Assay, Microscopy, Electron Microscopy