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Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease


Excess lipid accumulation in peripheral tissues is a key feature of many metabolic diseases. Therefore, techniques for imaging and quantifying lipids in various tissues are important for understanding and evaluating the overall metabolic status of a research subject. Here we present a protocol that detects neutral lipids and lipid droplet (LD) morphology by oil red O (ORO) staining of sections from frozen tissues. The method allows for easy estimation of tissue lipid content and distribution using only basic laboratory and computer equipment. Furthermore, the procedure described here is well suited for the comparison of different metabolically challenged animal models. As an example, we include data on muscular and hepatic lipid accumulation in diet-induced and genetically induced diabetic mice. The experimental description presents details for optimal staining of lipids using ORO, including tissue collection, sectioning, staining, imaging and measurements of tissue lipids, in a time frame of less than 2 d.

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Figure 1: Neutral lipid accumulation in tissues from metabolically challenged mice.


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We want to thank S. Wittgren, E. Cortez and C. Mössinger for technical assistance. This study was supported by The Novo Nordisk Foundation, The Swedish Cancer Foundation, The Swedish Research Council, Torsten och Ragnar Söderbergs Stiftelser, The Peter Wallenberg Foundation for Economics and Technology, The Swedish Heart and Lung Foundation, Ludwig Institute for Cancer Research and Karolinska Institutet. C.E.H. was kindly supported by Frans Wilhelm och Waldemar von Frenckells Fond and by Wilhelm och Else Stockmanns Stiftelse.

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A.M. performed in vivo mouse experiments, ORO analyses and wrote the paper; C.E.H. optimized the protocol and commented on the paper; L.M. commented on the paper; U.E. commented on the paper; A.F. optimized the protocol, performed ORO analyses, assisted in in vivo mouse studies and wrote the paper. All the authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Annelie Falkevall.

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The authors declare no competing financial interests.

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Mehlem, A., Hagberg, C., Muhl, L. et al. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protoc 8, 1149–1154 (2013).

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