Goat anti rabbit igg h l horseradish peroxidase

Tumor-bearing mice were anesthetized with 1% pentobarbital and perfused with 4% paraformaldehyde. Tumors were captured and fixed in 4% paraformaldehyde, and paraffin sections of each group were serially sliced (6 μm thick) and subjected to immunohistochemical staining using the following antibodies: anti-CD31 antibody (Abcam, 1:500), anti-vimentin antibody (Proteintech, 1:100) and anti-E-cadherin antibody (Abcam, 1:500). The prepared slides were incubated overnight at 4°C and then incubated with goat anti-rabbit IgG (H+L) horseradish peroxidase (Beyotime Institute Biotechnology, Haimen, Jiangsu, People’s Republic of China; 1:100) for 40 min at 37°C. The antibody stainings were visualized with 3,3′-diaminobenzidine (DAB). A total of five areas were calculated per sample.

At the termination of the animal experiment, 4 µm sections of 4% paraformaldehyde-fixed paraffin-embedded tumor tissues were made. The slides were deparaffinized through a series of solutions (100% xylene through 100% ethanol to 100% water), followed by incubation with alpha-smooth muscle actin (α-SMA; CAF; 1:200 dilution; Proteintech, Wuhan, China), active TGF-β1 (1:500 dilution; Abcam, Cambridge, UK), CTGF (1:500 dilution; Abcam), or VEGF (1:250 dilution; Abcam) at 4°C overnight. Then, tumor sections were treated with goat anti-rabbit IgG (H + L) horseradish peroxidase (1:100 dilution; Beyotime, Haimen, China) at 37°C for 40 minutes. Immunoreactivity was visualized with 3,3′-diaminobenzidine (DAB) using microscope (Leica DM6000B).