Freshly blotted lung tissue samples were weighed on an aluminum f

Freshly blotted lung tissue samples were weighed on an aluminum foil, dried for 24 h at 95?, and reweighed. Difference in wet and dry tissue weights was calculated and expressed as wet dry ratio. Alveolar epithelial barrier function was evaluated by measuring Evans blue extravasation . Briefly, Evans blue was injected into the jugular vein of rats, 30 min before duct infusion. Lung tissue samples were obtained 6 h after duct infusion, sectioned and immersed in a formamide solution, homogenized for 2 min. After incubation at room temperature for 24 h, the suspension was centrifuged at 4000 g for 30 min. The amount of dye extracted was determined spectrophotometrically at 620 nm and calculated from a standard curve established with a known amount of Evans blue. Results were corrected by the wet dry lung tissue ratio and expressed as the dye content per dry weight of lung tissue . Western blotting analysis Western blotting analysis was performed as previously described . Total protein was separated from each sample by electrophoresis on a 4 20 SDS polyacrylamide gel and electroblotted onto polyvinylidene difluoride membranes.
Membranes were blocked in a blocking solution, incubated overnight with primary antibodies, and developed with a horseradish peroxidase conjugated secondary antibody diluted at 1:1000. Primary antibody was diluted as follows: claudin 4 at 1:100, claudin 5 at 1:100, and occludin at 1:300. The immune complexes were then visualized on X ray film using chemiluminecent HRP substrate Kinase Inhibitor Library . Additional immunoblots were performed using GAPDH antibody as the primary antibody to evaluate equal loading. Immunohistological analysis Lung tissue sections were dewaxed in graded alcohols, and washed with tap water. Endogenous peroxidase activity was blocked inhibitor chemical structure with 3 H2O2, and antigen was retrieved with microwave in 0.01 mol L citrate buffer. The sections were then washed with phosphate buffered saline . Mouse anti rat claudin 4 and claudin 5, and rabbit anti rat occludin polyclonal antibodies were diluted at 1:100 and incubated overnight at 4?. The sections were washed 4 times with PBS, 5 min once.
Power vision two step histostaining reagent was used for detection of claudin and occludin expression. All sections were developed using diaminobenzidine and counterstained with hematoxylin. The appropriate induction of pancreatitis associated lung injury was demonstrated by histology and elevated serum amylase activity . Lung injury was characterized by pulmonary PF-02341066 kinase inhibitor edema, leukocyte in?ltration, and alveolar collapse. Pulmonary pathological scores and serum amylase activity were significantly lower after treatment with emodin. Pulmonary edema was evaluated by measuring the water content in lung tissue samples and expressed as wet dry ratio, which was significantly decreased after treatment with emodin .

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