Mouse anti collagen type 1

The transduced keloid tissue explants were then fixed with 10% formalin, paraffin-embedded, and cut into 5-μm-thick sections. Representative sections were stained with Masson’s trichrome or Picrosirius red, and then examined by light microscopy. For immunohistochemical staining, keloid tissue explants sections were incubated at 4 °C overnight with mouse anti-Wnt3a (Abcam), rabbit anti-TGF-β1 (Abcam), mouse anti-collagen type-I (Abcam), mouse anti-collagen type-III (Sigma), mouse anti-elastin (Sigma), mouse anti-fibronectin (Santa Cruz Biotechnology), rabbit anti-MMP-9 (Abcam) primary antibody, and then incubated at room temperature for 20 min with the Dako Envision™ Kit (Dako, Glostrup, Denmark) as a secondary antibody. Diaminobenzidine/hydrogen peroxidase (Dako) was used as the chromogen substrate. All slides were counterstained with Meyer’s hematoxylin. The expression levels of Wnt3a, TGF-β1, collagen type-I, collagen type-III, elastin, and fibronectin were semi-quantitatively analyzed using MetaMorph^® image analysis software (Universal Image Corp., Buckinghamshire, UK). Results are expressed as the mean optical density of six different digital images.

Prepared normal dermal spheroids were then fixed with 10% formalin, paraffin-embedded, and cut into 5 μm-thick sections. Representative sections were stained with Picrosirius red, and then examined by light microscopy. For immunohistochemical staining, spheroid sections were incubated at 4 °C overnight with rabbit anti-TGF-β1 (Abcam), mouse anti-collagen type-I (Abcam), mouse anti-collagen type-III (Sigma), mouse anti-elastin (Sigma), or mouse anti-fibronectin (Santa Cruz Biotechnology) primary antibody, and then incubated at room temperature for 20 min with the Dako Envision™ Kit (Dako, Glostrup, Denmark) as a secondary antibody. Diaminobenzidine/hydrogen peroxidase (Dako) was used as the chromogen substrate. All slides were counterstained with Meyer’s hematoxylin. The expression levels of TGF-β1, collagen type I, collagen type III, elastin, and fibronectin were semi-quantitatively analyzed using MetaMorph® image analysis software (Universal Image Corp., Buckinghamshire, UK). The results are expressed as the mean optical density of six different digital images.