Alexa fluor 555 donkey anti mouse secondary antibody

MMA embedded sections were cut at 5μm thickness and stained with hematoxylin-eosin (H&E) and Movat Pentachrome. Immunoflourescent staining of endothelial cells was performed by labelling against CD31 (Dako Corp., Via Real - USA). Samples were initially incubated in 0.1% Triton X for 20 minutes and rinsed with PBS. The stent half was then subsequently exposed overnight at 4 °C to anti-CD31 (Dako Corp., Via Real – USA; dilution 1:20). The antibody reaction was visualized with an Alexa Fluor 555 donkey anti-mouse secondary antibody (Life Technologies, Carlsbad, CA dilution 1:150). DAPI (Life Technologies, Carlsbad - USA) was used as the nuclear counter stain. Selected cross-sections from rabbit iliac arteries were also stained with antibodies against RAM-11 (Dako Corp., Via Real – USA) to identify macrophages and foam cells.

Two millions HeLa cells were seeded in a 10 cm diameter dish, left untreated, irradiated (8 Gy), or intoxicated with CDT (1μg/ml) for the indicated periods of time. Cells were then detached with 2.5 mM EDTA in PBS. Four hundred thousand cells were incubated with the primary antibody (diluted 1:50 in PBS) on ice for 45 minutes. As positive control for integrin activation, cells were incubated with the primary antibody in PBS without Ca²⁺ and Mg²⁺ containing 2 mM MnCl₂ as previously described [31 (link)]. As negative control, cells were incubated with isotype-matched immunoglobulins. After washing in PBS, cells were incubated with the Alexa Fluor 555 donkey anti-mouse secondary antibody (Life technologies) for 45 minutes on ice. Flow cytometry analysis was performed using a FACSCalibur (BD Bioscience, San Jose, CA, USA). Data from 1x10⁴ cells were collected and analyzed using the CellQuest Pro software (BD Bioscience).
The following monoclonal antibodies were used: the pan anti-integrin β1 4B7R (R&D Systems) and HUTS-21 (BD Bioscience) that recognizes the activated form of integrin β1 [31 (link)].