Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins.
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.
Ohsaki
Yuki
Y
Department of Anatomy and Molecular Cell Biology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, 466-8550, Nagoya, Showa, Japan.
Maeda
Takashi
T
Fujimoto
Toyoshi
T
D000273:Adipose Tissue / Q000378:metabolism / Q000473:pathology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004072:Digitonin / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D050356:Lipid Metabolism; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D000070780:Perilipin-2; D000070796:Perilipin-3; D011257:Pregnancy Proteins / Q000737:chemistry / Q000378:metabolism; D012503:Saponins / Q000737:chemistry; D012995:Solubility; D016707:Tissue Fixation / Q000379:methods*; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C000602982:PLIN2 protein, human; C038196:PLIN3 protein, human; D000070780:Perilipin-2; D000070796:Perilipin-3; D011257:Pregnancy Proteins; C484168:RAB18 protein, human; D012503:Saponins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D004072:Digitonin