Immunohistochemical Analysis of Adipose Tissue

After fixation, AT was embedded in paraffin as described previously (22 (link), 29 (link)). At 4°C sections were incubated overnight with primary antibodies anti-Mac-2 (1:1000; Cedarlane; Burlington, ON, Canada; CL8942AP) and anti-PerilipinA (1:200; Abcam; Cambridge, UK; ab3526). Next, fluorochrome-conjugated secondary antibodies were applied for 1 h at room temperature (1:200; Invitrogen; Waltham, MA, USA). For nuclear staining DAPI (1:10,000; Thermo Fischer Scientific; Waltham, MA, USA; 62248) was used. For control stainings, same routines without primary antibodies were applied. A confocal Leica SPE microscope (Leica; Wetzlar, Germany) was used for image acquisition. Quantification of adipocyte size, interstitial macrophages and CLS density were performed semi-automatically with cellSens Software (Olympus; Hamburg, Germany) as described previously (30 (link)). H&E stainings were performed following standard routines (29 (link)).