Image pro plus 6.0 image analysis

The paraffin tissue sections were dewaxed in xylene and rehydrated in gradient alcohol. Then, the sections were washed with distilled water, and endogenous peroxidase was blocked by 3% hydrogen peroxide. The heat-mediated antigen retrieval was performed with citrate buffer at pH 6 before starting the IHC staining protocol. Then, the sections were blocked for 60 min with 5% goat sera. Indirect immunoperoxidase staining of tissue sections was performed, and the samples were incubated overnight with primary antibodies against Ki-67, ICAM-1, (Cambridge, MA, USA), eNOS, activated caspase-3 (Sigma-Aldrich, USA), and ROCK1 and ROCK2 (Proteintech, USA) at 4°C. Then, the tissue sections were washed with phosphate-buffered saline (PBS, 0.01 M), incubated with secondary antibodies, and visualized with diaminobenzidine (DAB, brown color, ZSGB-BIO, Beijing, China) and hematoxylin counterstaining under a microscope. The intensity of positive staining was measured by IOD/area with Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Silver Spring, MD).

Immunoperoxidase staining for p-SAPK/JNK, DNMT1, p65, EZH2 proteins were performed on all tissues from representative subjects (control, PPI-treated groups). Briefly, the antigen was retrieved by citric acid buffer (pH 6.0) and then immersed in 3 % H₂O₂ to inhibit endogenous peroxidase activity, followed by incubation in 5 % bovine serum albumin to block nonspecific binding. The sections were then incubated with primary antibodies against p-SAPK/JNK (1:100), p65 (1:100), EZH2 (1:50), DNMT1 (1:100) at 4 °C overnight and then incubated with biotinylated secondary antibody (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Beijing, China) for 10 min. Detection was made using the 3,3 -diaminobenzidine according to the instructions (DAB kit, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. China). Pictures were taken under 200× magnification. The immunostaining was evaluated by Image-Pro Plus6.0 image analysis software (Media Cybernetics, lnc. Sliver Spring, MD, USA) in at least five random high-power fields.