H550s microscope

We used an Olympus BX41 microscope with a CCD 1300QDS digital camera to obtain digital images from G-banded and C-banded karyotypes, which were analyzed using the GenASIs software v. 7.2.7.34276. A Nikon H550S microscope with a DS-Qi1Mc digital camera captured the FISH images, which were analyzed using Nis-Elements software. The karyotypes were organized according to literature^{57 (link)}. The final images were edited using Adobe Photoshop software v. 22.1.1.

Cell-seeded scaffolds from the three chosen time points (7, 14, and 21 days after proliferation period) were fixed in 10% v/v neutral buffered formalin overnight at RT. After fixation, scaffolds were dehydrated in graded ethanol series, immersed in a toluole bath for 5 min, and then embedded in paraffin at 55 °C for 60 min. After blocking, paraffin cubes were cut transversely into 6 μm thick sections using a Leica RM 2235 rotary microtome. Selected sections, from equal distances throughout the whole scaffold, were heated at 60 °C for 30 min, deparaffinized, hydrated, and then stained with Hematoxylin and Eosin (H&E). Sections were dehydrated and covered. Similar protocol was also used for Alcian Blue staining (solution: 1% w/v pH = 2.5, adjusted with 3% glacial acetic acid of quality level 300, Sigma-Aldrich). After 21 days in culture, semiquantitative evaluation of collagen development was performed by staining the regenerated ECM in scaffolds with Indian ink. Ink has the appropriate chemical and physical properties to stain collagen within tissues [51 (link),56 ]. PBS was mixed with black Indian ink (2:1) for 2 min and then gently applied to the scaffolds with a 22-gauge needle. Stained scaffolds remained in RT for 5 min to equilibrate and were then immersed in PBS 3 times. Scaffolds were then placed on slides and examined with a Nikon H550s microscope.

We used an Olympus BX41 microscope and a CCD 1300QDS digital camera to obtain digital images from G-banded and C-banded karyotypes, which were analyzed using the GenASIs software v. 7.2.7.34276. The Nikon H550S microscope with a DS-Qi1Mc digital camera captured the FISH images, which were analyzed using the Nis-Elements software. The karyotypes were organized according to the literature^{83 (link)}. The final images were edited using Adobe Photoshop CS6 software.

Chromosomal preparations were obtained from the bone marrow in the field (Ford & Harmerton, 1956). As our sample is a female, the definition of the X chromosome was made by comparing it with the literature. The following techniques were applied, with adaptations: G‐banding (Sumner et al., 1971), C‐banding (Sumner, 1972), Ag‐NOR staining (Howell & Black, 1980), and FISH (Fluorescence In Situ Hybridization) with telomeric probes (All Human Telomere Probe: Oncor, P5091) (Nagamachi et al., 2013) and 18S rDNA probes (Hatanaka & Galetti, 2004). Images of classic cytogenetics were obtained using an Olympus BX41 microscope (bright field/phase) with a digital CCD 1300QDS camera and analyzed using the SpectraView software (Applied Spectral Imaging). Images of FISH were obtained using a Nikon H550S microscope and analyzed using Nis‐Elements software. The images were edited using the Adobe Photoshop CS4 program.