For Mander's Overlap Coefficient (MOC) analysis z-stack images of S2 cells or larval motor neurons were generated at 160× and deconvolved using a constrained iterative algorithm that utilizes a theoretical point-spread function as previously described (Chang et al., 2013).
Degree of RyR-VDAC overlap was quantified with Mander's coefficient analysis (JaCop, ImageJ software).
These data have been further validated by colocalization analysis, showing the significant decrease of Manders' overlap coefficient in DM1 siRNA-transfected myotubes to the control value.
We further confirmed these data by performing voxel by voxel co-localization analysis on 3D images to generate a Mander's Overlap Coefficient (MOC), which is a quantitative measure of co-localization (Chang et al., 2013).
Second, the pathway crosstalk analysis was based on the scores measured by Jaccard Coefficient (JC) and Overlap Coefficient (OC).
Blind quantification of immunocolocalization degree by Mander's coefficient analysis revealed an age-dependent reduction of the fraction of RyR overlapping with VDAC (m1) without significant differences in either RyR or VDAC total fluorescences.
Mander's coefficient analysis of SR and mitochondria immunolabelling indicates that, while most RyR fraction (>75%) overlaps with mitochondrial VDAC in young cardiomyocytes, this interaction is reduced to <60% in old cardiomyocytes.
The first and last terms are normalized by a technique known as overlap coefficient [27].
The overlap coefficient was set to 0.5.
