Multifluorescence confocal microscopy: application for a quantitative analysis of hemostatic proteins in human venous valves.

Confocal laser scanning microscopy is commonly used to visualize and quantify protein expression. Visualization of the expression of multiple proteins in the same region via multifluorescence allows for the analysis of differential protein expression. The defining step of multifluorescence labeling is the selection of primary antibodies from different host species. In addition, species-appropriate secondary antibodies must also be conjugated to different fluorophores so that each protein can be visualized in separate channels. Quantitative analysis of proteins labeled via multifluorescence can be used to compare relative changes in protein expression. Multifluoresecence labeling and analysis of fluorescence intensity within and among human venous specimens, for example, allowed us to determine that the anticoagulant phenotype of the venous valve is defined not by increased anticoagulant expression, but instead by significantly decreased procoagulant protein expression (Blood 114:1276-1279, 2009 and Histochem Cell Biol 135:141-152, 2011).

Mechanisms of compensatory beta-cell growth in insulin-resistant rats: roles of Akt kinase.

The physiological mechanisms underlying the compensatory growth of beta-cell mass in insulin-resistant states are poorly understood. Using the insulin-resistant Zucker fatty (fa/fa) (ZF) rat and the corresponding Zucker lean control (ZLC) rat, we investigated the factors contributing to the age-/obesity-related enhancement of beta-cell mass. A 3.8-fold beta-cell mass increase was observed in ZF rats as early as 5 weeks of age, an age that precedes severe insulin resistance by several weeks. Closer investigation showed that ZF rat pups were not born with heightened beta-cell mass but developed a modest increase over ZLC rats by 20 days that preceded weight gain or hyperinsulinemia that first developed at 24 days of age. In these ZF pups, an augmented survival potential of beta-cells of ZF pups was observed by enhanced activated (phospho-) Akt, phospho-BAD, and Bcl-2 immunoreactivity in the postweaning period. However, increased beta-cell proliferation in the ZF rats was only detected at 31 days of age, a period preceding massive beta-cell growth. During this phase, we also detected an increase in the numbers of small beta-cell clusters among ducts and acini, increased duct pancreatic/duodenal homeobox-1 (PDX-1) immunoreactivity, and an increase in islet number in the ZF rats suggesting duct- and acini-mediated heightened beta-cell neogenesis. Interestingly, in young ZF rats, specific cells associated with ducts, acini, and islets exhibited an increased frequency of PDX-1+/phospho-Akt+ staining, indicating a potential role for Akt in beta-cell differentiation. Thus, several adaptive mechanisms account for the compensatory growth of beta-cells in ZF rats, a combination of enhanced survival and neogenesis with a transient rise in proliferation before 5 weeks of age, with Akt serving as a potential mediator in these processes.