Then the thorax was opened and the lungs removed. The left lung was fixated with intratracheal 4% formaldehyde, and immersed in the same fixative. The right lung was frozen at ?80 C for subsequent analysis. In additional selleckchem Bortezomib animals, a bronchoalveolar lavage was performed and the protein content of the bronchoalveolar lavage fluid measured. Histological studies After fixation, the left lung was included in paraffin, and a standard hematoxilin eosin staining was performed in three lung sections, separated by 1 mm intervals. A previ ously Inhibitors,Modulators,Libraries described score was used to quantify the severity of lung damage. To measure the neutrophil infiltration of lung tissue, additional sections were immunostained using an anti myeloperoxidase antibody.
The number of myeloperoxidase positive cells in three randomly chosen high power fields per section was counted and averaged. Activation of Nuclear factor ��B was measured by counting the percentage of posi tive nuclei in histological sections immunostained with an anti p65 antibody. Biochemical Inhibitors,Modulators,Libraries measurements The right lung was homogenized in a lysis buffer with a protease inhibitors cocktail. The sam ples were centrifuged and the supernatants collected and stored. Protein content was measured using a BCA assay. Nuclear extracts from lung tissue were prepared as pre viously described. Briefly, frozen tissues were homog enized in a cold buffer, centrifuged and the pellets resuspended in the same buffer with 0. 1% Triton X 100. After incubation, samples were centrifuged and the nuclear pellets resuspended in a buffer containing 20 mM Tris pH8, 25% glycerol, 0.
4 M NaCL, 1. 5 mM MgCl2, 0. 2 mM EDTA, 0. 5 mM DTT and protease inhibitor cocktail. After incu bation, the nuclear extracts were finally centrifuged and the supernatants collected and frozen at ?80 C. Gene expression was studied Inhibitors,Modulators,Libraries by quantitative PCR as de scribed. Total RNA was extracted from tissue using Trizol and isopropanol precipitation. Using this RNA, cDNA was synthesized Inhibitors,Modulators,Libraries and quantitative real time PCR carried out in triplicate for each sample. Expres sion of Cxcl2 and beta actin was measured using Taqman probes. Relative expression was computed according to manufac turers instructions. For western blotting assays, samples were loaded in a 10% SDS polyacrilamide gel and electrophoresed.
The pro teins were then transferred to a nitrocellulose membrane and incubated with primary antibodies against p65, E selectin, Intercellular Inhibitors,Modulators,Libraries adhesion molecule 1 or beta actin. The binding of pri mary antibodies was detected by using a peroxidase linked secondary antibody and a chemoluminiscent reaction in a LAS 3000 camera. Actin was used as loading control. Matrix metalloproteinases 2 and ?9 were mea sured by gelatin zymography, as previously described. selleck kinase inhibitor IL 10 was quantified using an ELISA, following manufacturers instructions. Statistical analysis All the results are expressed as mean SEM.