Immunohistochemical and Immunofluorescent Analysis of Adipose Tissue

Formalin-fixed, paraffin-embedded SAT sections were used for IHC and IF investigations as described previously (12 (link), 28 (link)). Anti-HSP60 (Enzo LifeSciences, Inc., Lausen, Switzerland), anti-TNF-α (Abcam, Inc., Cambridge, MA, USA), and anti-IL-6 antibodies (Novus Biologicals, LLC, Littleton, CO, USA) were used for IHC. Quantification of the IHC data was performed using ImageScope software version 11.1 (Aperio, Vista, CA, USA) as previously reported (12 (link)). For IF staining, tissue sections were incubated with an Alexa Fluor^® 488-conjugated Anti-HSP60 antibody (Bioss Inc., Woburn, MA, USA). DAPI was used at 0.05% for nuclear staining. The sections were analyzed using a Zeiss LSM 710 confocal laser-scanning microscope, and fluorescent images of the representative areas of the adipose tissue were photographed using a × 40 objective.