Rabbit anti tgf β1

Sections were baked, dewaxed and hydrated as previously described. The antigen was then repaired by proteinase K (0.3 mg/ml; Merck KGaA) treatment at 37°C for 30 min. Endogenous enzyme activity was extinguished using 3% hydrogen peroxide for 10 min. Slices were blocked with goat serum (cat. no. 5425; Cell Signaling Technology, Inc.) at 37°C for 1 h and incubated with primary antibody overnight at 4°C [rabbit anti-TGFβ1, cat. no. 3709, 1:100; rabbit anti-phosphorylated (p)-Smad2/3, cat. no. #8828S, 1:200; Cell Signaling Technology, Inc.; rabbit anti-a disintegrin and metalloproteinase with thrombospondin motifs 4 (Adamts4), cat. no. #ab185722, 1:200; Abcam], incubated with secondary antibody for 1 h at room temperature [horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody, cat. no. #7074, 1:1,000; Cell signaling Technology, Inc.; goat anti-rabbit lgG/Cy3, cat. no. bs-0295G-Cy3; 1:1,000; Bio-synthesis Inc.]. 3′-Diaminobenzidine (DAB, Bio-synthesis Inc.) was added for 5 min and then 4′, 6-diamidino-2-phenylindole or hematoxylin was used to counterstain the nuclei at room temperature for 5 min. The slides were observed under a light or fluorescence microscope (magnification, ×40, ×100 or ×200).