After pressurization, skin specimens were collected and fixed with 10% formalin-buffered solution (Wako Pure Chemical Industries, Ltd.) and then embedded in paraffin blocks. Immunohistochemical staining for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and cleaved caspase-3 were performed for investigation of apoptotic skin, while Hematoxyline-Eosin (HE) staining was used to confirm the changes of the skin structure. In in vivo experiment, 1 and 4 weeks after implantation, three mice per group were euthanized using carbon dioxide inhalation. The implanted grafts (n = 6 per group) were harvested and fixed with 10% formalin-buffered solution (Wako Pure Chemical Industries, Ltd.). Paraffin Sects. 5-μm-thick of each specimen were subjected to HE staining and immunohistochemical staining for F4/80 and anti-CD31. Finally, histological sections were examined by NanoZoomer 2.0 HT imaging (Hamamatsu Photonics, Hamamatsu, Japan) at 40× magnification and analyzed using the NDP.view2 software program (Hamamatsu Photonics). Cell nuclei in 3 areas measuring 250 × 250 μm (at the center and on both sides 500 μm from the center) on HE sections at 1 and 4 weeks after implantation were counted and compared.
Ndp view2 software program
Manufactured by Hamamatsu Photonics
Sourced in Japan
The NDP.view2 software program is a digital imaging software developed by Hamamatsu Photonics. It is designed to provide users with a user-friendly interface for viewing and analyzing digital images captured using Hamamatsu's photonic products.
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4 protocols using ndp view2 software program
1
Histological Assessment of Skin Grafts
2
Histological Analysis of Implanted Grafts
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At one, two and eight weeks after implantation, four mice per group were euthanized using carbon dioxide inhalation. The implanted grafts (n = 8 per group) including the surrounding tissue (2 × 2 cm) and muscle underneath were harvested. Tissue specimens were cut axially at the center using a scalpel, fixed with 10% formalin-buffered solution (Wako Pure Chemical Industries, Ltd.), and then embedded in paraffin blocks. Paraffin sections 5-μm-thick from the central region of each specimen were subjected to HE staining, Azan staining and immunohistochemical staining for anti-vimentin and anti-CD31. After staining, histological photographs were taken and analyzed using a NanoZoomer 2.0 HT whole-slide imager with the NDP.view2 software program (Hamamatsu Photonics, Hamamatsu, Japan) at 40× magnification.
3
Quantifying Dermal Thickness in Skin Grafts
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The thickness of each graft after 8 weeks was evaluated on Azan-stained sections. Azan staining stains dermal collagen fibers a dark blue that can be differentiated from the connective tissue stained a light blue. The maximal dermal thickness of the right third, central third, and left third as well as that of each graft were estimated for these sections and compared. The cross-sectional area of the dermis also measured on Azan-stained sections at Week 8 using the NanoZoomer 2.0 HT whole-slide imager with the NDP.view2 software program (Hamamatsu Photonics).
4
Histological Analysis of Tissue Sections
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The tissue sections after MSI were stained for histological analysis with hematoxylin (Merck KGaA, Darmstadt, Germany) and eosin (Wako, Japan). The serial frozen sections were stained by TdT-mediated dUTP Nick End Labeling (TUNEL) [18] (link). The H&E staining after MSI was as follows: (1) fix in a 4% paraformaldehyde for 30 min, (2) wash in water for 5 min, (3) stain in hematoxylin for 5 min, (4) wash in water for 5 min, (5) dip in 1% HCl 70% ethanol for 2 s, (6) wash in water for 5 min, (7) (link) counterstain in eosin Y for 20 s, (8) wash and dehydrate in 100% ethanol for 5 min, (9) wash and dehydrate again in 100% ethanol for 5 min, (10) (link) dip in xylene for 5 min, and (11) (link) dip in xylene again for 5 min. The sections were covered with marinol (Wako, Japan), followed by a glass cover slide, and dried at room temperature. TUNEL-stained sections were created using the TUNEL stain kit (Merck MILLIPORE, Darmstadt, Germany) as directed by the manufacturer's protocol. Histological sections were scanned with a NanoZoomer 2.0 HT slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) at 40× magnification and observed using the NDP.view2 software program (Hamamatsu Photonics). HE-stained images were converted to black and white mode in 8-bit type grayscale by image J software (NIH, http://imagej.nih.gov/ij/, accessed on 14 October 2021).
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