Mouse monoclonal antibody against icam 1

Immunohistochemical staining was carried out with the Dako Envision System (Dako, Glostrup, Denmark). In brief, formalin-fixed, paraffin-embedded tissue sections were incubated overnight at 4° C with mouse monoclonal antibody against ICAM-1 (Santa Cruz, CA, USA) at 1:100 dilutions. Slides with no primary antibodies added served as negative controls.

The effects of triptolide on the expression of adhesion molecules, including ICAM-1 and VCAM-1, were determined using cell-based ELISA assay as previously described [20 (link)]. In brief, HUVECs in 96-well plates were fixed with 4% paraformaldehyde for 5 min and rinsed 3 times with PBS following treatment. The fixed cells were permeabilized with pre-chilled MeOH for 10 min at -4 °C, followed by blocking with PBS containing 1% BSA and 0.2% triton X-100 for 1 h. Subsequently, the cells were incubated with mouse monoclonal antibody against ICAM-1 or mouse monoclonal antibody against VCAM-1 (dilution, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C for 12 h. Following washing, the cells were incubated with FITC-conjugated anti-mouse secondary antibody (dilution, 1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. Incubation with PBS, instead of the primary antibodies, was used as a negative control. The optical density of each well was determined by the use of a SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 485/520 nm. The optical density of the negative control was subtracted as background from that of each well.