Rabbit anti tgf β1

Tissue sections were stained with hematoxylin and eosin (HE) and Masson’s trichrome (MT) stains. For immunohistological examination, the following primary antibodies were used: rat anti-NIMP-R14, a Ly-6G/-6C neutrophil marker (1:400; Hycult Biotech, Netherlands), rat anti-CD31 (1:30; Dianova GmbH, Hamburg, Germany), rat anti-F4/80 (1:200; Abcam plc), rabbit anti-TGF-β1 (1:800; Abcam plc) rabbit anti-αSMA (1:2000; Abcam plc), rabbit anti-Smad3 (1:1000; Gene Tex Inc., CA, USA), rabbit anti-phosphorylated Smad3 (p-Smad3) (1:8000; Rockland Immunochemicals Inc., Limeric, PA), rabbit anti-myoglobin (1:1000; Abcam plc), and rabbit anti-myogenin (1:200; Abcam plc). All were diluted with Antibody Diluent (DAKO Japan Inc., Tokyo, Japan). For the CD31, TGF-β1, myoglobin, and myogenin antibodies, antigens were activated by heating in 0.01 M citrate buffer (pH 6.0) for 5 min. Sections were incubated overnight with the primary antibody at 4 °C. The immunobound antibodies were detected using biotin-conjugated secondary antibodies in the N-Histofine Simple Stain Mouse MAX PO (Rat) detection system (Nichirei Biosciences Inc., Tokyo, Japan) and the EnVision kit (DAKO Japan Inc.), and the sections were developed by 3,3′-diaminobenzidine stain and counterstained with hematoxylin. Stained sections were observed under a microscope (Bio-Zero and BZ-X800; KEYENCE Co.).

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.

Primary antibodies used in this study included rabbit anti-SOX-2 (1:1000 dilution, Abcam, Cambridge, UK), rabbit anti-Nanog (1:1000 dilution, CST, Danvers, Massachusetts, USA), rabbit anti-OCT-4 (1:1000 dilution, Abcam), rabbit anti-pSmad2/3 (1:500 dilution, Abcam), rabbit anti-Snail (1:500 dilution, Abcam), rabbit anti-E-cadherin (1:500 dilution, Abcam), rabbit anti-TGF-β1(1:1000 dilution, Abcam), rabbit anti-N-cadherin (1:1000 dilution, Wanleibio), rabbit anti-Slug (1:1000 dilution, Wanleibio) and mouse anti-β-actin (1:1000 dilution, ZSGB-BIO, Beijing, China).

The left ventricles were homogenized in an ice-cold RIPA lysis buffer (Thermo Scientific, Waltham, MA) with a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). These samples were loaded in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were then incubated with rabbit anti-collagen 1 (1 : 5000, Abcam, Cambridge, MA, USA), rabbit anti-TGF-β1 (1 : 1000, Abcam), or rabbit anti-tubulin (1 : 5000, Millipore) antibodies overnight. After that, membranes were washed and incubated with goat anti-rabbit HRP-coupled secondary antibodies (1: 10,000). The results were visualized by an enhanced chemiluminescence detection system (PerkinElmer, Boston, MA, USA).

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.