Ipp 6.0 imaging software

Vascular smooth muscle cell migration was determined using Transwell chambers, with a Transwell membrane containing 8-μm pores (Costar; Corning) and coated with 10 μg/mL fibronectin, inserted into a 24-well plate. The lower chamber was filled with 600 μL of DMEM with 10% FBS, and VSMCs (1 × 10⁵ cells/well) in serum-free DMEM were placed in the upper chamber, followed by incubation with PBS or various concentrations of triptolide (0, 5, and 10 ng/mL) for 24 h. Infiltrated cells were fixed in 4% paraformaldehyde (Bioss, C01-06002) and stained with 0.1% crystal violet (Bioss, D10162). Migrated cells were photographed under an inverted microscope (LEICA DMI 4000B, Germany), and five random high-power fields (200× magnification) were selected for quantification of cell number using the IPP 6.0 imaging software (Media Cybernetics). VSMC-migration ability was expressed as the ratio of the number of migrated cells to that of control cells.

For IHC analysis, the cross-sections (4-μm thick) were deparaffinized and rehydrated, followed by incubation with antibodies against CD3 (Abcam, ab135372, 1:800), CD4 (Abcam, ab183685, 1:800), CD8 (Abcam, ab217344, 1:1000), and CD68 (Abcam, ab125212, 1:1000) at 4°C overnight. The samples were then stained with Goat Anti-rabbit IgG/HRP (Bioss, bs-0295G-HRP, 1:100) for 1 h at 37°C. Adventitial CD3+, CD4+, CD8+, and CD68+ cells were scored by cell counting using IPP 6.0 imaging software (Media Cybernetics) and expressed as cell number per vessel section (400× magnification).