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Keywords:

  • LPS;
  • Mrp2;
  • Bsep;
  • liver;
  • confocal immunofluorescence microscopy;
  • quantitative colocalization;
  • software

Abstract

Quantitative colocalization analysis is a powerful tool for reliable estimation of the colocalization of antigens. We employed it to determine the changes of colocalization of multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) in confocal immunofluorescence microscopy images of rat liver following lipopolysaccharide (LPS) administration. Samples were taken 2, 24, 48 hours, and 1 week after LPS challenge. Pearson's correlation coefficient (PCC), an overlap coefficient according to Manders' (MOC), and overlap coefficients k1 and k2 were used to explore changes of the degree of colocalization. In intact animals, confocal microscopy showed tight colocalization of Mrp2 and Bsep proteins exclusively at the bile canaliculi. High degree of colocalization was confirmed quantitatively. Injection of LPS resulted in the appearance of fuzzy-looking areas of fluorescence of both proteins around bile canaliculi 2 and 24 hours after administration and relocation of Mrp2 protein to the basolateral domain of hepatocytes at 48 hours. By 1 week, canalicular localization was restored morphologically. Quantitative colocalization analysis of canalicular regions showed a steady decrease of the degree of colocalization of Mrp2 and Bsep up to 48 hours with the slight increase of its value by 1 week. These findings demonstrate that Mrp2, in contrast to Bsep, is partially and reversibly relocated from canalicular to basolateral domain of hepatocytes after LPS challenge. Microsc. Res. Tech. 67:65–70, 2005. © 2005 Wiley-Liss, Inc.