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.

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PKN (protein kinase N; also called protein kinase C-related kinase (PRK-1)), is a serine/threonine protein kinase that is ubiquitously expressed in several organs, including the brain. PKN has a molecular mass of 120 kDa and has two domains, a regulatory and a catalytic domain, in its amino-terminals and carboxyl-terminus, respectively. Although the role of PKN has not been fully elucidated, previous studies have revealed that PKN is cleaved to a constitutively active catalytic fragment of 55 kDa in response to apoptotic signals. Hydrocephalus is a pathological condition caused by insufficient cerebrospinal fluid (CSF) circulation and subsequent excess of CSF in the brain. In this study, in order to elucidate the role of PKN in the pathophysiology of hydrocephalus, we examined PKN fragmentation in hydrocephalic model rats.
Hydrocephalus was induced in rats by injecting kaolin into the cisterna magna. Kaolin-induced rats (n=60) were divided into three groups according to the observation period after treatment (group 1: 3–6 weeks, group 2: 7–12 weeks, and group 3: 13–18 weeks). Sham-treated control rats, injected with sterile saline (n=20), were similarly divided into three groups. Spatial learning ability was estimated by a modified water maze test. Thereafter, brains were cut into slices and ventricular dilatation was estimated. Fragmentation of PKN was observed by Western blotting in samples collected from the parietal cortex, striatum, septal nucleus, hippocampus, and periaqueductal gray matter.
All kaolin-induced rats showed ventricular dilatation. Most of them showed less spatial learning ability than those of sham-treated controls. In most regions, fragmentation of PKN had occurred in a biphasic manner more frequently than that in controls. The appearance of PKN fragmentation in periaqueductal gray matter was correlated with the extent of ventricular dilation and spatial learning disability. These results revealed that PKN fragmentation was observed in rats with kaolin-induced hydrocephalus, models for chronically-damaged brain dysfunction, suggesting that persistent brain insult, such as apoptosis, had occurred in these models. PKN fragmentation could be a hallmark for evaluating morphological and functional damage of the hydrocephalus.

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The expression of intermediate filaments is sensitively reflected in cell function. To examine the involvement of keratin in a secretory function, 15 kinds of keratin (keratin-2, 3, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, 18, 19, 20) were detected immunohistochemically and immunoelectron microscopically in the rabbit duodenum. Four types of secretory cells existed in the rabbit duodenum: enteroendocrine cells and goblet cells in the epithelium and mucous and serous cells in the duodenal glands. Among the 15 kinds of keratin, keratin 20 was selectively expressed in all these secretory cells. However, localization of keratin 20 in the endocrine cells differed from that in three types of exocrine cells. In the enteroendocrine cells, keratin 20-containing filaments formed a juxtanuclear network from which they extended to the apical cell membrane. These filaments may play a role in intracellular signal transduction, since the apical cell membrane contains some receptors for binding a specific extracellular signal. In the exocrine cells, on the other hand, keratin 20-containing filaments existed just beneath the cell membrane. These filaments may play some role in maintaining cell shape, which is remarkably changed during the secretory cycle.

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