Quantitative colocalization analysis is an advanced digital imaging tool to examine antigens of interest in immunofluorescence images obtained using confocal microscopes. It employs specialized algorithms to estimate the degree of overlap of fluorescence signals and thus enables acquiring important new information not otherwise obtainable using qualitative approaches alone. As raw confocal images have high levels of background, they should be prepared to become suitable for reliable calculation of colocalization coefficients by correcting it. We provide concise theoretical basis of quantitative colocalization analysis, discuss its limitations, and describe proper use of the technique. The use of quantitative colocalization analysis is demonstrated by studying bile salt export pump and multidrug resistance associated protein 2 in the liver and major basic protein and platelet activating factor receptor antigens in conjunctiva. The review is focused on the applicability and correct interpretation of the results of colocalization coefficients calculations.

Colocalization is an important finding in many cell biological studies. This unit describes a protocol for quantitative evaluation of images with colocalization based on the calculation of a number of specialized coefficients. First, images of double‐stained sections are subjected to background correction. Then, various coefficients are calculated. Meanings of the coefficients and a guide to interpretation of their results indicating either presence or absence of colocalization are given. Success in colocalization studies depends on the quality of analyzed images, proper preparation of them for coefficients calculations, and correct interpretation of obtained results. This protocol helps to ensure reliability of colocalization coefficients calculations. Curr. Protoc. Cell Biol. 39:4.19.1‐4.19.16. © 2008 by John Wiley & Sons, Inc.

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. V V C 2005 Wiley-Liss, Inc.

The action of platelet-activating factor (PAF) toward the PAF receptor (PAF-R) plays an important role in inflammation. We employed immunohistochemistry and quantitative confocal immunofluorescence microscopy to examine the dynamic changes of PAF-R expression in the conjunctiva in response to PAF-induced conjunctivitis in Brown Norway rats within the first 2 h after topical administration of PAF. Instillation of PAF caused an alteration in pattern of recruitment of macrophages and eosinophils into the conjunctiva, as was visualized by immunohistochemical staining for the antigens ED 1 and ED 2 (markers of macrophages) and MBP (a marker of eosinophils). An increase in the number of PAF-R positive cells was alos detected. Quantitative colocalization analysis revealed ther strongest rise in the degree of PAF-R expression by macrophages within the first 6 h, whereas their infiltration increased throughout the period of observation. However, eosinophils showed a high degree of PAF-R expression during all 24 h of the experiment, although they infiltrated strongly only within the first 2 h. Thus, for the first time, the use of quantitative colocalization analysis software developed by us has reveal intrinsic details of the interaction of PAF anf PAF-R in conjunctivitis, findings not otherwise obtainable by using qualitative approaches alone. Our results provide a theoretical basis for a definition of the proper time-frame in which to prevent and control the infiltration of macrophages and eosinophils into the conjunctiva.

Chronic ethanol consumption elicits detrimental changes of liver metabolism. By employing a 12-week-long feeding regimen, we investigated the effects of chronic ethanol consumption on the expression and localization of bile salt export pump (Bsep), a major canalicular exporter of bile salts, and multidrug resistance protein 2 (Mrp2), a canalicular organic anion transporter, in the rat liver. RT-PCR, confocal immunofluorescence microscopy, immunoblotting, and quantitative colocalization analysis were used to examine their gene and protein expression, and changes in the distribution of antigenic sites. Bsep mRNA was upregulated, while Mrp2 mRNA responded by downregulation. In agreement with mRNA, the expression of Bsep protein increased, while the expression of Mrp2 protein responded with a decrease, suggesting that the expression of both of them is transcriptionally regulated. Confocal immunofluorescence microscopy showed disruption of the colocalization of Bsep and Mrp2 proteins at the hepatocyte canalicular membrane and their relocation intracellularly. Quantitative colocalization analysis of Bsep and Mrp2 proteins revealed a steady decrease in the degree of colocalization and Mrp2 expression, indicating that although the properties of both transporters are affected, Mrp2 is altered more. These findings provide evidence that ethanol alters Bsep and Mrp2 canalicular transporters in the rat liver, at both the mRNA and protein levels. Mrp2 shows deeper involvement. Eight weeks appears to be a critical time point in this process.