2.3. Morphology and Immunohistochemistry Analysis

The left kidney tissues were immobilized in 10% neutral formalin solution for 24 hours and then underwent gradient dehydration, transparency, paraffin immersion, paraffin embedding, and hematoxylin and eosin (HE) staining. To assess histopathologic changes in the kidney, 10 high magnification (×200) was randomly selected in each sample and scoring method was based on Tervaert et al. [18]. For Masson dyeing, the histological sections underwent dewaxing and dehydration; then the tissues were stained with hematoxylin and Ponceau red liquid dye acid complex. The tissues were then directly stained with aniline blue liquid and 1% acetic acid after soaking in 1% phosphomolybdic acid solution. For the analysis of fibrotic area, 10 high magnification (×200) was randomly selected and photographed in each sample. Collagen observed as the blue area represents the extent of the fibrosis, and the areas were calculated by image analysis software (Image-Pro Plus 6.0) for semiquantitative score.

For immunohistochemistry staining, the activity of endogenous enzymes was blocked by 3% hydrogen peroxide after the kidney sections were repaired by microwave antigen for 15 min; then the tissue was blocked by goat serum for 15–20 min. Primary antibodies used in this research contained alpha-smooth muscle actin (α-SMA, 1 : 200, Abcam, ab5694), fibronectin (FN, 1 : 100, Proteintech, 15613-1-AP), and PAR-1 (1 : 50, Abcam, ab32611) overnight at 4°C. After washing with phosphate-buffered saline (PBS), the sections were covered with secondary antibody for 1h and then accordingly 3,3′-diaminobenzidine (DAB) and hematoxylin were added. 10 high magnification (×400) was randomly selected in each sample. The percentage of positive staining area was calculated by image analysis software (Image-Pro Plus 6.0) for semiquantitative score.