ARTICLE |
Correspondence to: Dawn L. Brasaemle, Dept. of Nutritional Sciences, 96 Lipman Drive, Rutgers, State University of New Jersey, New Brunswick, NJ 08901. E-mail: Brasaemle@AESOP.Rutgers.edu
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Summary |
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The study of proteins associated with lipid droplets in adipocytes and many other cells is a rapidly developing area of inquiry. Although lipid droplets are easily visible by light microscopy, few standardized microscopy methods have been developed. Several methods of chemical fixation have recently been used to preserve cell structure before visualization of lipid droplets by light microscopy. We tested the most commonly used methods to compare the effects of the fixatives on cellular lipid content and lipid droplet structure. Cold methanol fixation has traditionally been used before visualization of cytoskeletal elements. We found this method unacceptable for study of lipid droplets because it extracted the majority of cellular phospholipids and promoted fusion of lipid droplets. Cold acetone fixation is similarly unacceptable because the total cellular lipids are extracted, causing collapse of the shell of lipid droplet-associated proteins. Fixation of cells with paraformaldehyde is the method of choice, because the cells retain their lipid content and lipid droplet structure is unaffected. As more lipid droplet-associated proteins are discovered and studied, it is critical to use appropriate methods to avoid studying artifacts.
(J Histochem Cytochem 51:773780, 2003)
Key Words: lipid droplets, oil droplets, oil bodies, adipocytes, triacylglycerol, immunofluorescence, microscopy, light microscopy, chemical fixation, adipophilin, perilipin
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Introduction |
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Lipid droplets are found in many different organisms and across many different tissues and cell types. Although the lipid droplets within adipocytes in the adipose tissue of animals are the largest and most easily observed, these specialized structures have been found across the biological kingdom and are now characterized as ubiquitous components of most types of cells (
To date, only a few lipid droplet-associated proteins have been identified. Adipophilin is a ubiquitously expressed protein in all mammalian cell types and is found only in lipid droplets and in no other subcellular compartment (
Immunofluorescence microscopic techniques have been used to study the localization of proteins in cells for many years, resulting in the development of an assortment of standard methods. Given the recent expansion of research initiatives investigating lipid droplet-associated proteins, an evaluation of these methods is now imperative. Chemical fixation is used to preserve cell or tissue structure so that the ensuing imaging methods accurately capture the localization of cell components that existed in the cell at the time of fixation (
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Materials and Methods |
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Materials
Ham's F12 medium, Dulbecco's minimal essential medium (DMEM), and trypsin were purchased from Mediatech (Herndon, VA). Fetal bovine serum, oleic acid, fatty acid-free bovine serum albumin, saponin, paraformaldehyde, goat IgG, monoclonal anti-ß-tubulin antibody clone TUB 2.1, and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG were purchased from Sigma (St Louis, MO). Lissaminerhodamine-conjugated goat anti-rabbit IgG was purchased from Jackson Immunoresearch (West Grove, PA). Goat anti-rabbit AlexaFluor 488 and Bodipy 493/503 were purchased from Molecular Probes (Eugene, OR). The BCA Protein Assay Kit was purchased from Pierce Chemical Company (Rockford, IL). Five percent ammonium sulfate-impregnated silica gel thin-layer chromatography plates were purchased from Analtech (Newark, DE). Methanol and acetone were purchased from Fisher Scientific (Pittsburgh, PA).
Cell Culture
CHO fibroblasts were maintained in Ham's F12 medium and 3T3-L1 cells were maintained in DMEM. Media for both cell lines were supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained at 37C in a 5% CO2 atmosphere. The cells were grown in culture flasks and dishes from Corning Life Sciences (Acton, MA). Cells for microscopy experiments were grown on glass coverslips in 100-mm culture dishes.
The differentiation of 3T3-L1 cells to adipocytes was accomplished by incubating confluent monolayers of cells in DMEM supplemented with 10% fetal bovine serum, 0.5 mM isobutylmethylxanthine, 10 µg/ml insulin, and 10 µM dexamethasone with fresh medium changes every 24 hr for 72 hr, followed by DMEM supplemented with 10% fetal bovine serum with medium changes every 24 hr for an additional 72 hr (
Lipid loading of CHO fibroblasts was achieved when the cells reached confluence by supplementing cell culture medium with 400 µM oleic acid complexed to fatty acid-free bovine serum albumin (6:1 moles of oleate:mole of albumin) (
Fixation of Cells
Methanol Fixation.
Methanol was stored at -20C before use. The culture medium was removed and the cells on coverslips were washed twice with PBS. The cells were incubated with cold methanol (
Acetone Fixation.
Acetone was stored at -20C before use. The culture medium was removed from the cells, and the cells were incubated with cold acetone for 10 min (
Paraformaldehyde Fixation.
Medium was removed from the cells, which were then washed twice with PBS. Cells were incubated with 3% paraformaldehyde in PBS for 20 minutes at room temperature (
Immunofluorescence Microscopy
All incubations were done in antibody diluent (PBS with 0.1 mg/ml saponin and 0.5 mg/ml goat IgG); all washes were done with PBS. Fixed cells were incubated for 45 min at RT in antibody diluent with 0.2 M glycine (
Protein Assay
To test the effects of chemical fixation on the protein content of cells, total protein mass was assessed after the fixation protocols. Densely subconfluent CHO cells were incubated with fixatives and rinsed with PBS, as described above. For control (no fixative) conditions, cells were washed six times with PBS before harvest to mimic the wash conditions of the fixed cells. Cells were harvested by scraping into PBS. Protein content of the collected cells was measured by the bicinchoninic acid method (
Lipid Extraction and Analysis
To test the effects of chemical fixation on the lipid content of cells, total lipids were extracted from cells after the fixation procedures in 2:1 chloroform:methanol (
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Results |
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We tested three fixation procedures that have been used before microscopy of lipid droplets and their associated proteins for their effects on cellular lipid content and lipid droplet structure. Cold acetone and cold methanol fixatives have traditionally been effective for subsequent viewing of cytoskeletal elements. Paraformaldehyde crosslinks proteins to preserve structure in cells and has been used for many applications. CHO cells were grown to confluence and then loaded with oleic acid complexed to bovine serum albumin for 24 hr to increase the storage of triacylglycerols in lipid droplets (
Paraformaldehyde Fixation Preserves Lipid Droplet Structure Most Effectively; Methanol Fixation Facilitates Fusion of Lipid Droplets
Paraformaldehyde-fixed CHO cells showed the most consistent staining of adipophilin surrounding lipid droplets of all sizes (Fig 1A1C). Methanol-fixed CHO cells generally showed stronger staining of neutral lipids than acetone or paraformaldehyde-fixed cells (Fig 1). However, methanol-fixed cells appeared to lack adipophilin staining on the smallest lipid droplets, and the adipophilin staining appeared discontinuous around the larger lipid droplets so that there were visible gaps in the surface staining (Fig 1D and Fig 1F). In general, the droplets of the methanol-fixed cells looked larger than those of paraformaldehyde-fixed cells, suggesting that fusions of the lipid droplets may have occurred within these cells. To quantify the observation that the methanol-fixed cells had larger lipid droplets than the paraformaldehyde-fixed cells, lipid droplets from 50 cells per treatment, fixed by either the methanol or the paraformaldehyde protocols, were sized and counted. The results showed that methanol-fixed cells have significantly more lipid droplets larger than 2.5 µm and fewer droplets smaller than 1 µm per cell compared to paraformaldehyde-fixed cells (Fig 2).
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Acetone Fixation Extracts the Core Neutral Lipids
Acetone-fixed CHO cells had minute structures that stained for adipophilin but not for neutral lipid (Fig 1G1I). The staining pattern for adipophilin was fragmented at the surfaces of the structures and also appeared in globular shapes that were not representative of the circular and continuous adipophilin staining pattern observed in the paraformaldehyde-fixed cells (Fig 1A1C).
Methanol Fixation of 3T3-L1 Adipocytes Confirms Lipid Droplet Fusion Events
To confirm our initial findings from experiments with CHO cells, the methanol and paraformaldehyde fixation procedures were tested on differentiated 3T3-L1 adipocytes, which have much larger lipid droplets. Pre-adipocytes were seeded onto glass coverslips, grown to confluence, and then treated with isobutylmethylxanthine, dexamethasone, and insulin to induce differentiation. Fully differentiated adipocytes were fixed and stained for immunofluorescence microscopy with antibodies raised against perilipins, the major lipid droplet-associated proteins in adipocytes, and with Bodipy 493/503 to detect neutral lipids. Methanol-fixed cells showed larger droplets, obvious fusion of lipid droplets with evidence of partially fused droplets clearly visible, and less defined perilipin staining at the edges of the droplets, with higher levels of diffuse perilipin staining in the droplet interiors (Fig 3D3F) when compared to 3T3-L1 adipocytes fixed with paraformaldehyde (Fig 3A3C). Paraformaldehyde-fixed adipocytes had smaller droplets, no visible fusion of droplets, clearer distinct staining of neutral lipid, and heavy continuous perilipin staining around all of the lipid droplets.
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Methanol and Acetone Fixation Procedures Were Most Effective Before Visualization of Microtubules
Because the cold methanol and cold acetone procedures have been most frequently used to visualize cytoskeletal elements, CHO cells fixed by the three procedures were stained for tubulin, a cytoskeletal protein, and were observed with a fluorescent microscope. Visualization of microtubules was much clearer in cells fixed with cold methanol (Fig 4A) or cold acetone (Fig 4B) than with paraformaldehyde (Fig 4C). Tubulin fluorescence was weaker in paraformaldehyde-fixed cells, and the captured images lacked clarity and definition.
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Relative to Unfixed Cells or Cells Fixed with Paraformaldehyde, Cells Fixed with Acetone Lacked the Majority of Neutral and Polar Lipid Content; Methanol-fixed Cells Lacked the Majority of Phospholipids
Because methanol and acetone are used routinely in lipid extraction procedures, we suspected that the visible alterations in lipid droplet structure were due to the extraction of droplet lipids. To determine the effect of the fixation procedures on the cellular lipid content, lipid analysis was performed on lipid-loaded CHO cells that were fixed by the three procedures. Four conditions were tested: unfixed control cells and cells fixed with either paraformaldehyde, cold methanol, or cold acetone. Total lipids were analyzed by quantitative thin-layer chromatography, and the lipid content was expressed relative to cellular protein content to control for differences in cell numbers due to the fixation procedures. We observed significant lifting of cells from the growth surface when cold acetone or cold methanol fixatives were used. To compare four separate experiments in which the initial total lipid content varied due to small differences in lipid loading conditions, all data were expressed as percentages relative to the unfixed control cells. Cells fixed with cold acetone had no detectable triacylglycerol and only 25% of the total phospholipid content compared to paraformaldehyde-fixed and control cells (Fig 5). Methanol-fixed cells were depleted of more than 80% of their phospholipid. Paraformaldehyde-fixed cells had levels of triacylglycerols and phospholipids comparable to the unfixed control cells.
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Discussion |
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The most important finding of this study is that paraformaldehyde is the most effective fixative to use for studying the structure of lipid droplets in cells. Cold methanol and cold acetone fixation procedures extracted lipids from the cells and altered the appearance of lipid droplets visualized by immunofluorescence microscopy. Cold acetone fixation procedures quantitatively extracted cellular triacylglycerol, thus eliminating potential Bodipy 493/503 staining of droplets, while altering the staining pattern of adipophilin associated with the outer surfaces of lipid droplets in cultured CHO cells. Many of the lipid droplets displayed a collapsed appearance, possibly reflecting the removal of the core lipid. Cold methanol fixation, on the other hand, extracted the majority of phospholipids. Partial or complete fusion of lipid droplets was observed in most cells, and staining for adipophilin or perilipins on the lipid droplets of CHO cells or adipocytes, respectively, became weak and discontinuous. Paraformaldehyde was the least destructive fixative, yielding clear images of distinct spherical lipid droplets bounded by continuous rings of adipophilin or perilipin staining and with lipid content comparable to control cells. These findings demonstrate that the choice of fixative is critical in studies of lipid droplet structure, because the extraction of cellular lipids by the fixative may alter the appearance of surface-associated proteins and the shape and size of the droplet.
Cold acetone and cold methanol procedures were initially developed to study the structure of cytoskeletal elements in cells. From this study, it is clear that these fixation methods are superior to paraformaldehyde fixation for visualizing microtubules. The effectiveness of these methods in visualizing the cytoskeletal network is likely due to the removal of phospholipids from cell membranes, because these membranes hinder the access of antibodies to the cytoskeletal proteins.
Fixation of cells with cold methanol resulted in fusion of lipid droplets and a greater intensity of the staining of neutral lipids with Bodipy 493/503. Extraction of the surface monolayer of phospholipids on the surfaces of the lipid droplets probably facilitates the entry of the fluorophore into the neutral lipid core. Furthermore, removal of the surface phospholipids may remove a barrier to lipid droplet fusion. Because methanol fixation extracts almost all of the cellular phospholipids and alters the staining pattern of surface proteins on lipid droplets, this study raises the question of whether the visualization of proteins associated with membranous compartments other than the limiting phospholipid monolayer of lipid droplets, such as lysosomes, endocytic vesicles, Golgi, and endoplasmic reticulum, may also be adversely affected by methanol fixation procedures.
We hope that the results of this study will lead to the re-evaluation and standardization of fixation methods for the study of lipid droplet-associated proteins and lipid droplet structure. We have found that aldehyde fixation methods are more effective than alcohol fixation methods in preserving lipid content and lipid droplet structure. Furthermore, it would be useful to develop an adequate fixation procedure to permit the subsequent visualization of both lipid droplets and cytoskeletal elements.
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Acknowledgments |
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Supported by NIH R01 DK54797.
We thank Dr Susan Fried, Dr Anne Garcia, and Dr Richard Ludescher for critical review of the manuscript.
Received for publication November 6, 2002; accepted January 15, 2003.
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