Histofluid mounting medium

Adipose tissues were fixed overnight in buffered formaldehyde (10%) and embedded in paraffin and were prepared at the thickness of 5 mM. The sections were deparaffinized and dehydrated with three changes of xylene for 15 min each and two changes of 100% alcohol for 5 min each. Sections were stained with haematoxylin (Sigma-Aldrich) for 1 min and differentiated in 1% acid alcohol for 10 s before counter-stained in eosin–phloxine solution (Sigma-Aldrich) for 5–6 s. Sections were then cleared in three changes of xylene for 5 min each and mounted with histofluid mounting medium (Marienfeld-Superior, Germany). Cell size quantification was performed using software by calculating pixels of adipocytes on HE staining. For immunohistochemistry, sections were sequentially incubated with primary antibody UCP-1 (Abcam, 10983, rabbit polyclonal, 0.5 ug ml⁻¹) overnight and anti-rabbit secondary antibody (Cell Signaling Technology, 7074 s, 0.1 ug ml⁻¹) for 1 h at room temperature, followed by development with 3,3-diaminobenzidine solution (Sigma-Aldrich). The nuclei were counter-stained with haematoxylin. Two independent investigators blinded to sample identity performed the staining and analyzed the adipose tissue sections respectively.

Haematoxylin/eosin (H/E) staining was carried out as previously described [28 (link), 51 (link)]. Briefly, formalin-fixed paraffin-embedded tissue sections (5 μm) were deparaffinised by heating in a dry oven (15 min, 65 °C) followed by two consecutive 10 min incubations in xylene. Rehydration was achieved by subsequent washes in EtOH solutions with increasing content of deionized H₂O (dH₂O): twice in 100% EtOH, then once in 96, 70 and 50% EtOH, and finally in dH₂O. Sections were stained in Mayer’s haematoxylin solution (Merck) for 12 min, and then washed in running tap water (15 min). The sections were then incubated in 0.5% eosin Y solution with phloxine (Merck) (90 s). Subsequently, the sections were dehydrated in solutions with an increasing EtOH content: 70, 96 and 100% followed by washing in xylene. The slides were sealed with Histofluid mounting medium (Paul Marienfeld, Lauda-Königshofen, Germany). Microscopic evaluation of H/E samples was carried out by assessing 10 random fields of view in a ‘blind’ manner. The severity of each parameter—oedema, inflammatory cell infiltration, and acinar cell necrosis—was scored using a four-level grade [28 (link)].

Mouse liver tissue, epididymal adipose and brown adipose were fixed in 10% neutral-buffered formalin, embedded in paraffin and sectioned at 5 μm. H&E staining was performed using standard techniques.
Pancreatic tissues were harvested, embedded in Tissue-Tek, and ‘snapfrozen' in dry ice and ethanol and stored at −80 °C. Cryostat sections (10 μm in thickness) were prepared, air-dried and fixed in ice-cold acetone for 15 min. Sections were blocked with 5% goat serum, stained with anti-Insulin antibody (Santa Cruz Biotechnology, Dallas, TX, USA), mounted with histofluid mounting medium (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and analysed with an Olympus FV1000 microscope (Olympus, Shinjuku-ku, Tokyo, Japan). Images were acquired with Olympus Fluoview Version 2.1 software.