Uncoupling protein 1 (UCP1) is a thermogenic biomolecule expressed in the mitochondrial crista of adipocytes in the brown adipose tissue (BAT) of mammals [1] [2] [3] [4]. The biological functions of UCP1 include thermogenesis and antioxidative reactions that enable the conversion of free electrons for heat generation. Therefore, evaluation of the expression of UCP1 in the BAT may give an insight into energy homeostasis involving glucose and lipid metabolism, as well as oxidative reactions [5]. The involvement of periarterial and pericardial BAT in metabolic and arteriosclerotic disorders emphasizes the need to evaluate UCP1 function around the arteries and heart [6] [7] [8] [9]. Nevertheless, some difficulties exist in the quantitative evaluation of UCP1 expression in lipid-rich organs such as BAT around relatively restricted areas, such as the periarterial and pericardial regions of small experimental animals such as mice [10] [11]. A successful approach to this problem was shown by Cinti et al. using an immunoelectron microscope [12], and developed by other researchers using gold particles conjugated to secondary antibodies [13] [14] [15] [16] [17]; however, the numerical characteristics of the immunopositive reactions remain obscure.

In this study, we quantitatively evaluated the expression of UCP1 in the interscapular BAT of mice by immunohistochemical staining using a gold particle-conjugated secondary antibody and electron microscopy. In addition, we statistically compared the diameters and densities of high-intensity electron signals of gold particles in the mitochondrial area under staining conditions with or without primary or secondary antibodies.

The BAT exhibited a multilocular appearance with small fat droplets and abundant mitochondria along with UCP 1 expression in the cytoplasm (Supplementary Figures S1-S6). The expression site of UCP1 was confirmed in the inner

membrane of the mitochondrial crista by immunohistochemical staining and electron microscopy (Figure1 and Figure2 and Supplementary FigureS6(b)). Immunoelectron images of UCP1 revealed the immunoreactive clusters of gold particles immunochemically conjugated to the UCP1 antibody. Next, gold particles were significantly identified using the diameter indicated by the manufacturer in the univariate logistic regression analysis (Figure5). A quantitative comparison of the patterns of the immunoreactive clusters between the polyclonal IgG anti-UCP1 antibody and monoclonal antibody revealed only a few differences in the diameter and density of the particles within the immunoreactive

clusters (Figure 3 and Figure 4). Statistical evidence underlying the immunoreactive clusters was evaluated by calculating the diameter and density of the immunoreactive particles on the mitochondrial inner crista (Figure 3 and Figure 4). Polyclonal and monoclonal IgG anti-UCP1 antibodies appeared to function similarly as carriers of immunoreactive clusters. The monoclonal IgG anti-UCP1 antibody exhibited clearer immunoreactive clusters, although they were not significantly different from those exhibited by the polyclonal antibody.

The immunoelectron microscopy method was previously established [12]. The immunoreactive clusters of gold particles conjugated to the polyclonal IgG

anti-UCP1 antibody observed in the mitochondria clearly indicated the site of UCP1 expression in the mitochondria of rats where the protein expression was upregulated upon exposure to cold. Previous studies have quantitatively analyzed immunoreactive clusters of gold particles using immunoelectron microscopy [13] [14] [15] [16] [17]. The accuracy of the diagnostic evaluation using the diameters of immunoreactive gold particles in the logistic regression analysis revealed the significant effective scores for sensitivity and specificity as compared with those of UCP1 antibody and the negative control. The obtained diameter of the immunopositive particles appeared to reflect the manufacturer’s data sheet for the diameter of the gold particles. Therefore, we established that verification of the diameters of immunoreactive gold particles could indicate the reliability of the method. Although a few reports have compared the immunohistochemical responses of UCP1 antibody in the BAT between monoclonal and polyclonal IgGs using electron microscopy, our study demonstrated the statistical efficacy of the anti-UCP1 antibody of both polyclonal and monoclonal IgGs using the diameters and densities of the immunoreactive gold particles in the immunoelectron staining of the mouse BAT.

Although our study has a limitation owing to the lack of a stimulus reaction test such as cold exposure or catecholamine beta-3 receptor agonist treatment where UCP1 might be induced on the inner membrane of the mitochondrial crista of the BAT [1] [2] [3] [20] [21], we demonstrated a significant threshold such as the mean diameter of the gold particle product between immunopositive reactions and nonspecific staining in immunoelectron microscopy. In the future, we intend to clarify the induction of UCP1 expression in the inner membrane of the mitochondrial crista of the BAT in mammals in response to changes in physiological or pathophysiological conditions using immunohistochemical staining and electron microscopy.

We thank Shuji Tatsukawa, Aiko Yasuda, and Hiroaki Kawazato for advice on electron microscopy, Yukari Gotoh for advice on light microscopy, and Keisuke Ina and Yoshihisa Fujikura for advice on the organization of this study. We would also like to thank the reviewers of this paper for their detailed feedback.

The authors declare no conflicts of interest regarding the publication of this paper.

Dong, X.M., Chiba, S., Shimada, T. and Hamada, F. (2022) A Statistical Evaluation of Uncoupling Protein 1 in the Limited Area of Brown Adipose Tissue by Immunoelectron Microscopy. Computational Chemistry, 10, 121-137. https://doi.org/10.4236/cc.2022.103006

For secondary antibodies in the light microscopic analysis, a peroxidase affinipure goat anti-rabbit IgG (111-035-144, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in combination with an EnVision System (anti-rabbit IgG-HRP conjugated, Dako Inc., Copenhagen, Denmark) and a Texas Red-affinipure goat anti-rabbit IgG (111-075-045, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in combination with 4’,6-diamidino-2-phenylindole dihydrochloride solution (DAPI) (28718-90-3, Dojindo Laboratories, Kumamoto, Japan) were used for immunohistochemical and immunofluorescence staining in the light microscopic observations, respectively. For the development of the physical method (physical development), AgNO₃ (Wako Pure Chemical Industries, Ltd., Osaka, Japan), crown ether/dibenzo-18-crown-6 ether (Sigma-Aldrich, St. Louis, MO, USA), and hydroquinone (Wako PureChemical Industries, Ltd., Osaka, Japan) were used. Next, a solution of distilled water 0.1% Kernechtrot (Chroma-Gesellschaft Schmid GmbH & Co, Nordrhein-Westfalen, Germany) and 5% aluminum sulfate (Wako PureChemical Industries, Ltd., Osaka, Japan) were used for nuclear staining in the physical development. Then, hematoxylin (Hx61057849, Merck KGaA, Darmstadt, DE) and eosin (E6003, Sigma-Aldrich, St. Louis, MO, USA) were used for general staining, and 150 mg/kg pentobarbital (Somnopentyl, Kyoritsu Seiyaku Corp., Tokyo, Japan) was applied for deep anesthesia before sample collection.

Fluorescence microscopy (blue in 461 nm, red in 610 nm) examinations were conducted using Eclipse 50i (Nikon Co., Ltd., Tokyo, Japan) and BX60 (Olympus Co., Ltd., Tokyo, Japan), respectively.

Samples that were fixed overnight were dehydrated with ethyl alcohol before embedding in paraffin. Next, coronal serial sections were cut with a thickness of 10 µm and fixed onto MAS-coated glass slides (Matsunami Glass Ind. Ltd., Osaka, Japan). Sections were deparaffinized in xylene, immersed in methanol with H₂O₂ for 30 min at 4˚C, and then hydratedin a series of descending ethanol concentrations before rinsing in running tap water for 5 min. Subsequently, the sections were incubated in 1% BSA in PBS (BSA–PBS; 0.01 M phosphate buffer + 0.15M NaCl, pH 7.2) for 30 min at room temperature (RT) and then incubated with rabbit polyclonal IgG anti-UCP1 primary antibody (1/1000) overnight at 4˚C. Serial sections were rinsed in BSA-PBS for 30 min at RT and incubated in a peroxidase affinipure goat anti-rabbit IgG secondary antibody (1/2000) for 1 h at RT before subjecting them to the EnVision System (for 2 h) and DAB visualization (for 10 min) at RT to detect UCP1 immunoreactivity (UCPi). Then, all sections were counterstained with hematoxylin. The specificity of UCP1 antibody was confirmed by performing the same procedure with rabbit polyclonal IgG (1/2000) instead of the primary rabbit polyclonal antibody to UCP1.

The physicalPhysical development is a method for intensifying the immunohistochemical reaction product [1]. The prepared coronal serial sections incubated with the rabbit polyclonal IgG anti-UCP1 primary antibody (1/1000) and rinsed in BSA-PBS similar to immunohistochemical staining were subsequently subjected to immunogold silver staining treatment. The serial sections were incubated in the 6-nm gold-conjugated goat anti-rabbit IgG antibody (1/40) for 1 h at RT, rinsed in 0.01 M PB (pH 7.4) four times for 30 min, and washed with distilled water for 10 s. Next, the physical developer was prepared by mixing distilled water with 10% AgNO₃ and 1.1 mM crown ether (solution A) and 0.2 M citric acid buffer (pH 3.45) with 30 mM hydroquinone (solution B). The immediately prepared physical developer was applied to the serial sections for 28 min at 23˚C. After rinsing in running tap water, the serial sections were counterstained with 0.1% Kernechtrot in 5% aqueous aluminum sulfate, rinsed in running tap water, and dehydrated in an ascending ethanol series (more details can be found in our previous report) [22].

The dehydrated sample sections were incubated in 1% BSA in PBS (BSA–PBS; 0.01 M phosphate buffer + 0.15 M NaCl, pH 7.2) for 30 min at RT and then incubated with rabbit polyclonal IgG anti-UCP1 (1/1000) overnight at 4˚C. Then, the serial sections were rinsed in BSA–PBS for 30 min at RT before incubation with Texas Red-affinipure goat anti-rabbit IgG secondary antibody (1/2000) for 1 h at RT in darkness. Then, the sections were rinsed in BSA-PBS for 30 min at RT, after which 1 µg/mL of DAPI was applied.

The collected samples were cut into very fine pieces, fixed in 50% Karnovsky’s fixative [23] for 10 min at 4˚C, rinsed with 0.1 M cacodylate buffer (pH 7.4), and post-fixed in 2% OsO₄ (0.1 M cacodylate buffer with 1% potassium ferrocyanide) for 2 h at 4˚C. The specimens were dehydrated in an ascending ethanol wash series and embedded in epoxy resin. Ultrathin sections were cut using an ultramicrotome, mounted on copper grids, and stained with methanolic uranyl acetate and lead citrate. Ultrathin sections were evaluated by Transmission electron microscopy (TEM) at an accelerating voltage of 80 kV [24].