A Statistical Evaluation of Uncoupling Protein 1 in the Limited Area of Brown Adipose Tissue by Immunoelectron Microscopy

Abstract

Uncoupling protein 1 (UCP1) expressed by the brown adipose tissue (BAT) in the mitochondrial crista acts as a homeostatic thermogenerator of eutherians. The evaluation of UCP1 expression in the BAT offers significant scientific insight, especially in studies targeting limited areas such as the periarterial and pericardial regions of small experimental mammals. However, the negligible amount of this adipose tissue would render the general quantitative evaluation of the protein unreliable because of lipid contamination and low protein concentration. To address this problem, we quantitatively evaluated UCP1 expression in the mitochondrion of the mouse interscapular BAT using immunoelectron microscopy and immunohistochemical studies using a combination of primary and secondary antibodies in scheme A (rabbit anti-UCP1 IgG/gold particle-conjugated goat anti-rabbit IgG), B (rabbit IgG/gold particle-conjugated goat anti-rabbit IgG), C (rabbit anti-UCP1 IgG/gold particle-unconjugated goat anti-rabbit IgG), and D (rabbit IgG/gold particle-unconjugated goat anti-rabbit IgG). Scheme A shows the immunopositive reaction of obvious gold particles in the mitochondrial area, whereas other procedures revealed less distinctive reactions. The distinctive gold particle immunoreaction comprised electrical high-density spots with a mean diameter of >5 nm. However, in scheme B, the electrical high-density spots were scattered outside the mitochondrion and were significantly smaller than 4 nm; schemes C and D demonstrated few immunoreactions. Logistic regression analysis between schemes A and B showed that the threshold diameter of the electrical high-density spots measuring >5 nm indicated a true positive immunoreaction to anti-UCP1 antibody specifically in the mitochondrial area. Minor statistical difference was observed in the primary anti-UCP1 antibody between polyclonal IgG and monoclonal antibodies. Therefore, immunoelectron microscopy might be useful for evaluating negligible protein expression in some limited areas, such as UCP1 expression in the BAT of small experimental animals.