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Methods Mol Biol. 2017;1594:73-92. doi: 10.1007/978-1-4939-6934-0_5.

Microscopic Analysis of Lysosomal Membrane Permeabilization.

Author information

1
Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
2
Core Facility Microscopy Unit, Medical Faculty, Linköping University, SE-581 85, Linköping, Sweden. vesa.loitto@liu.se.

Abstract

Lysosomes and lysosomal proteases have been found to participate during several forms of cell death pathways including apoptosis. A critical step in the mediation of apoptotic signaling is the release of cathepsins to the cytosol, a process known as lysosomal membrane permeabilization (LMP). In this chapter, we describe immunofluorescence detection of LMP in cell cultures stained for cathepsin B and LAMP-2 using three confocal techniques namely laser scanning, spinning disk, and aperture correlation spinning disk confocal to obtain images. Image analysis is performed using Huygens software for deconvolution. LMP results in a decrease in the fraction of cathepsin B colocalizing with LAMP-2, which is quantified through Manders' colocalization coefficient. Analysis of the images obtained by the three techniques show the same trend but the magnitude of the decrease differs due to the axial resolution. The observations emphasize the use of highest possible resolution when determining colocalization.

KEYWORDS:

Cathepsin B; Confocal microscopy; Image analysis; Immunocytochemistry; LAMP-2; Lysosomal membrane permeabilization

PMID:
28456977
DOI:
10.1007/978-1-4939-6934-0_5
[Indexed for MEDLINE]

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