4.8.2. Confocal Imaging

Cells were pre-stained with Cytopainter prior to cell seeding for 3D visualization of cell distribution. Cells were trypsinized, counted, and stained using CytoPainter Cell Proliferation Stain Deep Red (Abcam, ab176736) following manufacturer's protocol. Sca ffolds were seeded as above and imaged after 1 day of culture using a Nikon Confocal A1 + microscope at LBIC. The sca ffold was imaged by taking advantage of the autofluorescence, using 488 nm excitation.

#### 4.8.3. Fixation, Para ffin Embedding, and Sectioning

At selected time points sca ffolds were rinsed in PBS and fixated in 4% formaldehyde (VWR; Radnor, PA, USA) for 1 h, and subsequently dehydrated immediately (70% ethanol 1 h, 95% ethanol 1 h, 99.5% ethanol 30 min, 1:1 ethanol:xylene 15 min, xylene 30 min) or stored in PBS at 4 ◦C. Two changes of para ffin incubation at 60 ◦C (1 h and 30 min) were followed by embedding into para ffi n blocks, from which 4 μm thick sections were produced. Repopulated sca ffolds were processed in their sca ffold holder to ensure stretched morphology, and the center was punched out with a biopsy punch prior to embedding.
