Biology Reference
In-Depth Information
CHAPTER
Associated Proteins
12
Lydia-Ann L. S. Harris, James R. Skinner, and Nathan E. Wolins
Center for Human Nutrition, Washington University School of Medicine, St. Louis,
Missouri, USA
Imaging of Neutral Lipids
and Neutral Lipid
CHAPTER OUTLINE
Introduction and Rationale....................................................................................... 214
12.1 Materials........................................................................................................214
12.2 Methods .........................................................................................................215
12.2.1 General Considerations ............................................................... 215
12.2.2 Cell Lines .................................................................................. 215
12.2.2.1 Cell Growth and Density..................................................... 216
12.2.3 Microscopy ................................................................................ 217
12.2.3.1 Optimizing Antibody Concentration..................................... 217
12.2.4 Labeling Droplets with Lipophilic Fluorochromes........................... 217
12.2.5 Fluorescent Fatty Acids............................................................... 218
12.2.6 Testing for Optical Bleed Through and Cross-Reactivity.................. 218
12.2.7 Antibody Staining Protocol .......................................................... 219
12.2.7.1 Fat Droplets in Dispersed Muscle Fibers............................. 221
12.3 Discussion......................................................................................................221
12.3.1 Validation of Signals ................................................................... 221
12.3.2 Placing Fat Droplets in Intracellular Context ................................. 223
Summary ................................................................................................................ 224
Acknowledgments ................................................................................................... 224
References ............................................................................................................. 225
Abstract
Intracellular fat droplets are large and have a distinct morphology, which makes their
imaging at the light level simple and informative. We detail how to image the fat
droplet core by metabolic labeling with fluorescent fatty acids or lipophilic fluoro-
chromes. Further, we describe the use of indirect immunostaining to image fat drop-
let proteins and fat cores in the same field. We also address the use of appropriate
