In situ RT-PCR detection of inducible nitric oxide synthetase gene expression in lung during endotox

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Oblectlv To detect the Iocation of inducible nitlic rnoxide synthetase (iNOS) protein and mRNA in lung rnduring endotoxemia in rabbits.rnMethods blotting was perfomed before,rn 1 hour and 5 houm after the intravenous administration rnof lipolysaccharide (LPS) in rabbits. Immunornhistonbical analysls (IA), in situ hybridization and in rnsitu ireverse transcfipfiorl-polymerase chain reaction (in rnsltoJ RT-PCR) were also pedormed in lung sections.rnResults iNOS mRNA expression was found using rnNodhem blotting in lung 5 hours after LPS injection,rnwhile It was not found in contrei. Tbe positive stain was rnfound only in macrophages in lung 5 hours after LPS rninljecfion by standard hybddization and IA; while by in rnsitu RT- PCR, tbe amplifation products were found in rnmacrophaoes, aJlway epitheiial oells, vascular rnendotbeliei cells, smooth muscle cells and leukocytes,rnin addition to macrophages distributed abundantly rnthriygout the lung. The signal was absent in control rnor samples.rnCencluslons Using an in situ RT-PCR technique,rniNOS expressson was riot only observed in rnmacriogaqss also in many other kinds of cells in rnybg during endotoxemla in rabbits. This suggests that rnn situ RT-PCR ls much more sensirtive than in situ rnhybridization, and can be usad to examine gones with rnIow expreseion.
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