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Methods Enzymol. 2014;535:93-102. doi: 10.1016/B978-0-12-397925-4.00006-7.

Mild fixation and permeabilization protocol for preserving structures of endosomes, focal adhesions, and actin filaments during immunofluorescence analysis.

Author information

1
Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria.
2
Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria. Electronic address: lukas.a.huber@i-med.ac.at.

Abstract

Intracellular membrane trafficking is a highly dynamic process to sort proteins into either the recycling or degradation pathway. The late endosome is a major component of this endosomal biogenesis toward degradation by the lysosome. The endocytotic system is spread throughout the cytoplasm, and vesicle motility is achieved by multiple proteins including Rabs, motor proteins, and cytostructural elements. The subcellular localization of the late endosome is distributed from the accumulation in the perinuclear region toward the cell periphery. Using immunofluorescence methods combined with live-cell microscopy, we want to show that the preservation of the peripheral late endosomal compartment can be successfully achieved by two different techniques. On one hand, we compare two different widely used permeabilization methods: Triton X-100 and saponin. Comparing live-cell microscopic pictures of the same cell with immunofluorescences after fixation and permeabilization revealed improved results by the use of saponin. On the other hand, we present here a protocol of mild fixation to preserve peripheral structures like focal adhesion in combination with endosomes and actin filaments.

KEYWORDS:

Actin; Endosomes; Fixation; Focal adhesions; Immunofluorescence; Permeabilization; Saponin

[Indexed for MEDLINE]

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