Nunc labtek 2 chamber system

Freshly collected adipose tissue was fixed overnight in 10% formalin. Tissue was then washed three times in PBS/0.1 M glycine and permeabilized with 0.2% Triton-X for 15 min before blocking in PBS/1% BSA. Neutral fatty acids were stained with BODIPY^493/503 (2 μg/ml; Life Technologies, Carlsbad, CA, USA), and actin filaments were stained with phalloidin–AlexaFluor⁵⁴⁶, and nuclei stained with DAPI. Stained tissue was rinsed in PBS and mounted onto a coverslip using a Nunc Labtek II chamber system (Thermo Scientific, Waltham, MA, UAS) with Prolong Gold. The stained tissue was then visualized with a Leica Inverted SP5X Confocal Microscope System (University of Michigan Medical School Biomedical Research Core Facility).

Minced breast adipose tissue was fixed overnight in 10% formalin. Tissue was then washed three times in PBS/0.1 mol/L glycine and permeabilized with 0.2% Triton-X for 15 minutes before blocking in PBS/1% BSA. With its nonpolar structure and long-wavelength absorption and bright green fluorescence, BODIPY^493/503 is commonly used as an adipocyte stain for neutral fatty acids and other nonpolar lipids. Here, adipocytes were stained with BODIPY^493/503 (2 μg/mL, Life Technology), and actin filaments were stained with phalloidin-AlexaFluor⁵⁴⁶, and DAPI to identify nuclei. Stained tissue was rinsed in PBS and mounted onto a coverslip using a Nunc Labtek II chamber system (Thermo Scientific) with Prolong Gold. The stained tissue was then visualized with a Leica Inverted SP5X Confocal Microscope System (University of Michigan Medical School Biomedical Research Core Facility) (Figure 1).