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Published on: June 21, 2013
Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins
Yuki Ohsaki1, Takashi Maeda, Toyoshi Fujimoto
1Department of Anatomy and Molecular Cell Biology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, 466-8550, Nagoya, Showa, Japan.
Insights
Proper cell fixation and permeabilization methods are crucial for accurately visualizing lipid droplet (LD) proteins using immunofluorescence microscopy. Incorrect protocols can lead to the loss of key proteins like ADRP, TIP47, and Rab18 from LDs.
Area of Science:
- Cell Biology
- Biochemistry
- Microscopy Techniques
Background:
- The number of identified lipid droplet (LD)-associated proteins is growing.
- Reproducibility issues exist regarding the localization of certain LD proteins.
Purpose of the Study:
- To investigate the impact of fixation and permeabilization methods on the immunofluorescence detection of LD proteins.
- To identify optimal protocols for reliable LD protein visualization.
Main Methods:
- Immunofluorescence microscopy was employed.
- Cells were fixed using formaldehyde or glutaraldehyde.
- Permeabilization was performed using Triton X-100, digitonin, or saponin.
- Specific LD proteins (ADRP, TIP47, Rab18) were targeted.
Main Results:
- Formaldehyde fixation with Triton X-100 failed to detect ADRP, TIP47, or Rab18 on LDs.
- Formaldehyde fixation with digitonin or saponin enabled visualization of these LD proteins.
- Glutaraldehyde fixation with Triton X-100 allowed ADRP detection.
Conclusions:
- The choice of fixation and permeabilization significantly affects LD protein detection.
- Some detergents may solubilize LD proteins, necessitating specific fixation or mild permeabilization for retention.
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.
