Anti perilipin

Adipose tissues were immediately placed in fixative (10% formalin) solution. Histological sections (7 μm) were cut from formalin-fixed paraffin-embedded tissue blocks. Tissue sections were stained with hematoxylin–eosin (H&E) under standard conditions. Immunohistochemical staining was performed using a biotin-free immunoenzymatic antigen-detection system (Abcam, Cambridge, UK). For immunofluorescence staining, the sections were incubated with a combination of anti-perilipin (Fitzgerald, MA, USA) and anti-F4/80 (Abcam) at 4 °C overnight. After incubation with the corresponding fluorochrome-conjugated secondary antibodies, the sections were mounted and visualized using an LSM510 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany). The adipocyte area in the selected fat tissue sections was measured using iSolution DT 36 software (Carl Zeiss).

After the induction to lipid accumulation, the fixed cells were permeabilized with 0.2% Triton X-100 (Merck Millipore, Darmstadt, Germany), followed by incubation with 0.2% monkey serum for 20 minutes to prevent the nonspecific binding of immunoglobulins. An overnight incubation at 4°C was performed with anti-ADRP (1:1,200; Fitzgerald, Acton, MA, USA) and anti-perilipin (1:500; Biorbyt, Cambridge, UK). For the anti-ADRP and anti-perilipin primary antibodies, the secondary antibody staining was performed using Alexa 488-conjugated anti-monkey IgG (Fitzgerald) and cyanin 3-conjugated anti-rabbit (Fitzgerald), respectively, for 1 hour at room temperature. The slides were mounted in a commercial Fluoromount Mounting Medium that contained 4′,6-diamidino-2-phenylindole for nuclear detection (Sigma) and were examined using fluorescence microscopy followed by deconvolution (Olympus, Shinjuku, Tóquio, Japão). The samples incubated without the primary antibodies but with the secondary antibodies were used as the negative controls.

Histological sections (6 μm) were cut from formalin‐fixed paraffin‐embedded tissue blocks and were stained with hematoxylin–eosin (H&E) under standard conditions. Immunohistochemical staining was performed using the DAKO Envision system (DAKO, Carpinteria, CA, USA). Sections were immunostained with antibodies against UCP1 (Sigma‐Aldrich, St Louis, MO, USA). Peroxidase activity was detected with 3‐amino‐9‐ethyl carbazole. The adipocyte area in selected fat tissue sections was measured using iSolution DT 36 software (Carl Zeiss, Oberkochen, Germany).
For immunofluorescence staining, frozen sections were stained with anti‐Siglec‐F (Abcam, Cambridge, UK) or anti‐CD206 (Abcam) antibodies at 4°C overnight. Immunofluorescence analyses were performed to identify macrophages and eosinophils by staining tissues with a combination of anti‐perilipin (Fitzgerald, MA, USA) with either anti‐F4/80 or anti‐Siglec‐F antibodies, respectively. After incubation with the corresponding fluorochrome‐conjugated secondary antibodies, the sections were mounted and visualized using an LSM510 confocal laser scanning microscope (Carl Zeiss) installed in the Center for University‐Wide Research Facilities (CURF) at Chonbuk National University.