Skip to main content
SpringerLink
Log in

Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins

  • Original paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

The number of proteins known to be associated with lipid droplets (LDs) is increasing. However, the reported distribution of a given protein in the LDs was, in some cases, found not reproduced by other groups. We report here that the choice of the fixation and permeabilization method is important in order to observe LD proteins using immunofluorescence microscopy. Formaldehyde fixation followed by treatment with Triton X-100, one of the most frequently used protocols for the immunolabeling of cultured cells, was not appropriate to label adipocyte differentiation-related protein (ADRP), TIP47, or Rab18 in LDs. Formaldehyde fixation followed by treatment with digitonin or saponin, allowed the visualization of all these proteins in LDs. When cells were fixed with glutaraldehyde, permeabilization by Triton X-100 could also be used for ADRP. These observations suggest that LD proteins are likely to be solubilized by some detergents, and strong cross-linkage to the surrounding protein matrix or mild permeabilization is necessary for their retention on the LD surface.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Barbero P, Buell E, Zulley S, Pfeffer SR (2001) TIP47 is not a component of lipid droplets. J Biol Chem 276:24348–24351

    Article  PubMed  CAS  Google Scholar 

  • Bozza PT, Yu W, Penrose JF, Morgan ES, Dvorak AM, Weller PF (1997) Eosinophil lipid bodies: specific, inducible intracellular sites for enhanced eicosanoid formation. J Exp Med 186:909–920

    Article  PubMed  CAS  Google Scholar 

  • Brasaemle DL, Barber T, Wolins NE, Serrero G, Blanchette-Mackie EJ, Londos C (1997) Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein. J Lipid Res 38:2249–2263

    PubMed  CAS  Google Scholar 

  • Carroll KS, Hanna J, Simon I, Krise J, Barbero P, Pfeffer SR (2001) Role of Rab9 GTPase in facilitating receptor recruitment by TIP47. Science 292:1373–1376

    Article  PubMed  CAS  Google Scholar 

  • Cole NB, Murphy DD, Grider T, Rueter S, Brasaemle D, Nussbaum RL (2002) Lipid droplet binding and oligomerization properties of the Parkinson’s disease protein alpha-synuclein. J Biol Chem 277:6344–6352

    Article  PubMed  CAS  Google Scholar 

  • Diaz E, Pfeffer SR (1998) TIP47: a cargo selection device for mannose 6-phosphate receptor trafficking. Cell 93:433–443

    Article  PubMed  CAS  Google Scholar 

  • DiDonato D, Brasaemle DL (2003) Fixation methods for the study of lipid droplets by immunofluorescence microscopy. J Histochem Cytochem 51:773–780

    PubMed  CAS  Google Scholar 

  • Fujimoto T, Kogo H, Ishiguro K, Tauchi K, Nomura R (2001) Caveolin-2 is targeted to lipid droplets, a new “membrane domain” in the cell. J Cell Biol 152:1079–1085

    Article  PubMed  CAS  Google Scholar 

  • Fukumoto S, Fujimoto T (2002) Deformation of lipid droplets in fixed samples. Histochem Cell Biol 118:423–428

    Article  PubMed  CAS  Google Scholar 

  • Gocze PM, Freeman DA (1994) Factors underlying the variability of lipid droplet fluorescence in MA-10 Leydig tumor cells. Cytometry 17:151–158

    Article  PubMed  CAS  Google Scholar 

  • Hayashi T, Su TP (2003) Sigma-1 receptors [sigma(1) binding sites] form raft-like microdomains and target lipid droplets on the endoplasmic reticulum: roles in endoplasmic reticulum lipid compartmentalization and export. J Pharmacol Exp Ther 306:718–725

    Article  PubMed  CAS  Google Scholar 

  • Harlow E, Lane D (1988) Antibodies. A laboratory Manual. Cold Spring Harbor Laboratory, New York, p 418

    Google Scholar 

  • Heid HW, Moll R, Schwetlick I, Rackwitz HR, Keenan TW (1998) Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases. Cell Tissue Res 294:309–321

    Article  PubMed  CAS  Google Scholar 

  • Litvak V, Shaul YD, Shulewitz M, Amarilio R, Carmon S, Lev S (2002) Targeting of Nir2 to lipid droplets is regulated by a specific threonine residue within its PI-transfer domain. Curr Biol 12:1513–1518

    Article  PubMed  CAS  Google Scholar 

  • Liu P, Ying Y, Zhao Y, Mundy DI, Zhu M, Anderson RG (2004) Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J Biol Chem 279:3787–3792

    Article  PubMed  CAS  Google Scholar 

  • Londos C, Brasaemle DL, Schultz CJ, Segrest JP, Kimmel AR (1999) Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells. Semin Cell Dev Biol 10:51–58

    Article  PubMed  CAS  Google Scholar 

  • Miura S, Gan JW, Brzostowski J, Parisi MJ, Schultz CJ, Londos C, Oliver B, Kimmel AR (2002) Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium. J Biol Chem 277:32253–32257

    Article  PubMed  CAS  Google Scholar 

  • Murphy DJ (2001) The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog Lipid Res 40:325–438

    Article  PubMed  CAS  Google Scholar 

  • Ohashi M, Mizushima N, Kabeya Y, Yoshimori T (2003) Localization of mammalian NAD(P)H steroid dehydrogenase-like protein on lipid droplets. J Biol Chem 278:36819–36829

    Article  PubMed  CAS  Google Scholar 

  • Ostermeyer AG, Paci JM, Zeng Y, Lublin DM, Munro S, Brown DA (2001) Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets. J Cell Biol 152:1071–1078

    Article  PubMed  CAS  Google Scholar 

  • Ozeki S, Cheng J-L, Tauchi-Sato K, Hatano N, Taniguchi H, Fujimoto T (2005) Rab18 localizes to lipid droplet and induces its close apposition to the endoplasmic reticulum-derived membrane. J Cell Sci 118:2601–2611

    Article  PubMed  CAS  Google Scholar 

  • Pol A, Luetterforst R, Lindsay M, Heino S, Ikonen E, Parton RG (2001) A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance. J Cell Biol 152:1057–1070

    Article  PubMed  CAS  Google Scholar 

  • Tauchi-Sato K, Ozeki S, Houjou T, Taguchi R, Fujimoto T (2002) The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition. J Biol Chem 277:44507–44512

    Article  PubMed  CAS  Google Scholar 

  • Umlauf E, Csaszar E, Moertelmaier M, Schuetz GJ, Parton RG, Prohaska R (2004) Association of stomatin with lipid bodies. J Biol Chem 279:23699–23709

    Article  PubMed  CAS  Google Scholar 

  • Wolins NE, Rubin B, Brasaemle DL (2001) TIP47 associates with lipid droplets. J Biol Chem 276:5101–5108

    Article  PubMed  CAS  Google Scholar 

  • Yamaguchi T, Omatsu N, Matsushita S, Osumi T (2004) CGI-58 interacts with perilipin and is localized to lipid droplets. Possible involvement of CGI-58 mislocalization in Chanarin-Dorfman syndrome. J Biol Chem 279:30490–30497

    Article  PubMed  CAS  Google Scholar 

  • Yu W, Bozza PT, Tzizik DM, Gray JP, Cassara J, Dvorak AM, Weller PF (1998) Co-compartmentalization of MAP kinases and cytosolic phospholipase A2 at cytoplasmic arachidonate-rich lipid bodies. Am J Pathol 152:759–769

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Ms. Kumi Tauchi-Sato, Dr. Jinglei Cheng, and Mr. Tetsuo Okumura for technical assistance. This work was supported by Grants-in-Aid for Scientific Research and the 21st Century COE Program “Integrated Molecular Medicine for Neuronal and Neoplastic Disorders” of the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government.

Author information

Authors and Affiliations

  1. Department of Anatomy and Molecular Cell Biology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, 466-8550, Nagoya, Showa, Japan

    Yuki Ohsaki, Takashi Maeda & Toyoshi Fujimoto

Authors
  1. Yuki Ohsaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takashi Maeda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Toyoshi Fujimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Toyoshi Fujimoto.

Additional information

The authors Yuki Ohsaki and Takashi Maeda have contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ohsaki, Y., Maeda, T. & Fujimoto, T. Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins. Histochem Cell Biol 124, 445–452 (2005). https://doi.org/10.1007/s00418-005-0061-5

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-005-0061-5

Keywords

Search

Navigation