Tissues were fixed in Bouin’s fluid, embedded in paraffin, and then 4 µm sections were stained with hematoxylin and eosin (HE). Cell size was examined using HE-stained adipose tissues. Four arbitrarily chosen fields of view (0.12 mm2/WAT or 0.03 mm2/BAT per field) were analyzed by the computer program, NIH Image (Image J 1.62, National Institutes of Health, Bethesda, MD, U.S.A.), to estimate the adipocyte size. Subsequently, the density and distribution of adipocytes were calculated.
For immunohistochemistry, a rabbit polyclonal anti-UCP1 antibody (1 µg/ml, Alpha Diagnostic Intl. Inc., San Antonio, CA, U.S.A.) was reacted with deparaffinized sections overnight at 4°C and visualized with 3,3′-diaminobenzidine tetrahydrochloride using a Histofine Simple Stain MAX-PO kit (Nichirei, Tokyo, Japan) as described previously [6 (link)]. For estimation of Ucp1-positive area, arbitrary fields of view were analyzed using the NIH Image as described previously [9 (link)].
For immunohistochemistry, a rabbit polyclonal anti-UCP1 antibody (1 µg/ml, Alpha Diagnostic Intl. Inc., San Antonio, CA, U.S.A.) was reacted with deparaffinized sections overnight at 4°C and visualized with 3,3′-diaminobenzidine tetrahydrochloride using a Histofine Simple Stain MAX-PO kit (Nichirei, Tokyo, Japan) as described previously [6 (link)]. For estimation of Ucp1-positive area, arbitrary fields of view were analyzed using the NIH Image as described previously [9 (link)].