Immunohistochemical Analysis of Adipose Tissue

Adipose tissues were fixed and processed for histological analysis, as previously described [7 (link)]. Paraffin sections (5-μm thick) were subjected to immunohistochemical analysis, as previously described [7 (link)]. The antibodies used for immunochemical detection were anti-UCP1 antibody (rabbit, 0.5 μg/ml, Alpha Diagnostic International), perilipin 1 (rabbit, 1:100, Cell Signaling), and tyrosine hydroxylase antibody (mouse, 1:400, Merck Millipore). Secondary antibodies used were goat anti-rabbit-Alexa Fluor 488 and goat anti-mouse-Alexa Fluor 594 (1:500, Invitrogen, Molecular Probes). IgG controls (normal rabbit IgG, Santa Cruz) were used as negative controls for IHC analysis, when the information on the concentration of primary antibodies was available (Additional file 1: Figure S1). Otherwise, the omission of primary antibody was used as a negative control. DAPI (Sigma) was used as a nuclear counter stain.