Dky f58 ccdjvc

An immunostaining assay revealed the presence of MUSE cells in nanofat-derived
ASCs at cellular passage 4. N-ASCs were seeded on a glass with a diameter of 24
mm in six-well plates in triplicates with complete medium. The wells were
incubated at 37°C and 5% of CO₂. After 24 h, cells were fixed with 4%
buffered formalin for 1 h at 4°C in the dark, washed three times with sterile
PBS 1×, and incubated with SEEA3 (FITC conjugate, 1:200 dilution) and CD105 (APC
conjugate, 1:200 dilution) antibodies in the dark at 4°C for 30 min. At the end
of the incubation with the antibodies, the glasses were washed with PBS, and
mounting medium containing DAPI was added. The slices were imaged with an
Olympus BX-51 microscope (Olympus) equipped with a digital camera (DKY-F58 CCD
JVC) at 60× objective. Images were prepared using Las-X software.

To evaluate the morphology of the Hy-Tissue Nanofat, SVF
whole-mount assay was performed, as reported in Busato et al.^{30 (link)}
The emulsion was swiped in a histological glass and stained with
toluidine blue (Sigma-Aldrich). All slides were examined under an Olympus BX-51
microscope (Olympus, Tokyo, Japan) equipped with a digital camera (DKY-F58 CCD
JVC, Yokohama, Japan). In addition, for a deeper morphological understanding,
the Hy-Tissue Nanofat-SVF was studied in scanning electron
microscopy (SEM). The sample was fixed with glutaraldehyde 2% diluted in 0.1 M
phosphate buffer (pH 7.4) for 2 h at 4°C and post-fixed in 1% osmium tetroxide
(OsO₄) diluted in 0.2 M potassium hexacyanoferrate for 1 h at
4°C. The samples were dehydrated in a graded concentration of ethanol, followed
by a critical point dryer (CPD 030, Balzers, Vaduz, Liechtenstein), mounted to
stubs with colloidal silver and sputtered with gold by an MED 010 coater
(Balzers), and examined with FEI XL30 scanning electron microscope (FEI Company,
Eindhoven, The Netherlands). All the morphological analyses were performed on
each sample collected from every patient.