- To keep the box humid, a wet paper towel was placed in the
closed box together with the coverslips on top of the Parafilm. - For evaluation of dot-like distribution that is a result of the
lipidated LC3-II form associated with the autophagosomes,
the pinhole needs to be set to 1 airy unit to visualize only a
single focal plane of the cell to eliminate falsification of the
results due to fluorescent signals in other focal planes. - In order to count and evaluate the number of dots per cell in an
unbiased manner in different treatments, it is crucial to always
set the same brightness, contrast, and threshold for all pictures
of the same experiment to ensure comparability. This needs to
be applied strictly when taking the pictures as well as during the
later analysis. - For statistical analysis of the counted dots per cell, at least
100 cells should be analyzed. For this purpose, the tile scan
tool of the confocal microscope can be employed to take rather
unbiased pictures of many cells at the same time. - It is important not to mistake an increase in the overall fluores-
cence intensity with the induction of autophagy. The
LC3-positive dots mark autophagosomes, while a diffuse cyto-
plasmic distribution represents the LC3-I form that is not
associated with autophagic membranes.
Acknowledgments
This work was supported by a grant (“Alterations of Neuronal
Connectivity”) from the IZKF Aachen of the Medical School of
the RWTH Aachen University to J.V. and by a grant from the
START program of the Medical School of the RWTH Aachen
University to M.D. This work was also supported by the “Immu-
nohistochemistry and Confocal Microscopy Facility,” a core facility
of the IZKF Aachen within the Faculty of Medicine at RWTH
Aachen University.References
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