Fluorescent detection of lipid droplets and associated proteins.
Most eukaryotic cells can store excess lipid in cytosolic lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets with nile red and BODIPY 493/503 are included. The differences in the spectral properties of these two lipophilic dyes and advantages of each are discussed. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet-associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology. Immunofluorescent detection of adipophilin, a broadly expressed, lipid droplet-associated protein, widely used as a marker for lipid droplet accumulation, is presented as an example. Finally, a simple protocol for enhancing lipid droplet accumulation through supplementation with excess fatty acid is included.
Listenberger
Laura L
LL
State University of New York at Stony Brook, Stony Brook, New York, USA.
Brown
Deborah A
DA
D000818:Animals; D015415:Biomarkers; D001896:Boron Compounds / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured / Q000737:chemistry / Q000648:ultrastructure; D003470:Culture Media / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry*; D005057:Eukaryotic Cells / Q000737:chemistry* / Q000648:ultrastructure; D005227:Fatty Acids / Q000378:metabolism / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D007202:Indicators and Reagents; D008055:Lipids / Q000032:analysis*; D008322:Mammals / Q000033:anatomy & histology; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence / Q000379:methods*; D010078:Oxazines / Q000032:analysis / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D000070780:Perilipin-2
C527198:4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; D015415:Biomarkers; D001896:Boron Compounds; D003470:Culture Media; D005227:Fatty Acids; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008055:Lipids; D008565:Membrane Proteins; D010078:Oxazines; D010455:Peptides; D000070780:Perilipin-2; C044808:nile red