Confocal sp5

All incubation and wash steps were performed using a BioShake iQ at 500 rpm. For washing, 50 μl of PBS were applied per well for 5 min. In vitro differentiated subcutaneous white adipocytes were permeabilized in TBS-T buffer (0.05% Tween) for 10 min and blocked with 2% BSA in PBS for 30 min and washed twice with PBS, respectively. Primary antibodies were applied for 1 h at RT in antibody diluent (PLA kit, Sigma, DUO92101). The following antibodies were used: anti-mouse insulin receptor (1:5, ThermoFisher CT-1), anti-rabbit integrin beta 1 (1:10, ThermoFisher, SA40-08), and anti-rabbit integrin beta 3 (1:40, ThermoFisher, SJ19-09). As a negative control, no primary antibody was used. The PLA assay was implemented as stated in the manufacturer's protocol, using the Duolink™ In Situ PLA® ProbeAnti-Rabbit PLUS, Anti-Mouse MINUS and the In Situ Detection Reagents Orange (Sigma, DUO92101). The staining solution had a final concentration of 25 μg/ml BSA, 1 μg/ml of DAPI (Thermo), and Lipitox 488 (HCS LipidTOXTM Green Neutral Lipid Stain (1:200, InvitrogenTM, H34775) in 1x saline-sodium citrate buffer. Cells were washed 3 times, and imaging was performed with a Leica confocal SP5. Quantification was performed by counting the dots per nuclei of adipocytes.

SCF, PGF, BAT, and liver were taken and fixed in 4% paraformaldehyde (PFA, Carl Roth, Germany) at room temperature (RT) for 24 h dehydrated in an ascending row of ethanol (70–100%) and xylene. Subsequently, tissues were embedded with paraffin (Leica, Germany) and cut into 2-μm sections using a microtome (Leica, Germany). Dissected tissue sections were stained with hematoxylin and eosin (H&E) as previously described [33 (link)] and Masson Trichrome staining. Quantification of lipid amount was morphometrically determined by automatic digital image analysis using the commercially available software Definiens Developer XD 2 (Definiens AG, Germany). Immunofluorescent staining was performed on paraffin sliced tissue sections, where antigen retrieval was achieved by boiling with citric acid (pH = 6) for 10 min at 800 W. After blocking with filtered 3% bovine serum albumin/phosphate-buffered saline (BSA/PBS) at RT for 1 h, slides were incubated with primary antibody (Fibronectin 1:50) at 4 °C overnight. After subsequent washing (3x PBS, RT, 5 min), the slides were incubated with the secondary antibody (anti rabbit 488 (1:200) and (DAPI, 1:5,000)), for 1 h at RT in the dark. Eventually, the slides were washed (3x PBS, RT, 5 min) mounted, cover-slipped, and dried for 1 h. Imaging was performed with a Leica confocal SP5.