Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Karina Garcia; Andr&#233; Alves; Teresa M. Ribeiro-Rodrigues; Fl&#225;vio Reis; Sofia Viana

doi:10.3791/65206

Journal

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Biochemistry

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Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

JoVE Journal
Biochemistry

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JoVE Journal Biochemistry

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

5,584 Views

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07:12 min

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June 02, 2023

DOI:

10.3791/65206-v

DOI:

10.3791/65206-v

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07:12 min

June 02, 2023

•

53 Views

Karina Garcia^1,2,3,4, André Alves^1,2,3, Teresa M. Ribeiro-Rodrigues^1,2,3, Flávio Reis^1,2,3, Sofia Viana^1,2,3,4

¹Institute of Pharmacology & Experimental Therapeutics & Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine,University of Coimbra, ²Center for Innovative Biomedicine and Biotechnology (CIBB),University of Coimbra, ³Clinical Academic Center of Coimbra (CACC), ⁴Polytechnic Institute of Coimbra,Coimbra Health School

Transcript

This imaging approach allows the three-dimensional reconstruction of hepatic lipid droplet deposition and overcomes the main disadvantages of classic histological staining that narrowed the successful discrimination between micro and macrovesicular steatosis. The assessment of microvesicular steatosis is when gold-standard histological stainings are used, both and Oil Red, due to disruption and occasional fusion of native lipid droplets. The pro-BODIPY offers an alternative to the routine protocols for microvesicular hepatic steatosis assessment due to its right hydrophobicity for a fast uptake and effective tagging of lipid droplets, being not only insensitive to the environment polarity, but also extremely selective.

This imaging approach encloses broader applications in combination with immunochemistry techniques, allowing the simultaneous detection of other subcellular antigens that may advance the study of lipid droplets’biogenesis and dynamics, underpinning micro and macrovesicular hepatic steatosis.

Summary

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.