Extended Data Fig. 4: Analysis of two-color STORM images.
From: Mosaic composition of RIP1–RIP3 signalling hub and its role in regulating cell death
a, Representative histogram of localization precision of CF 568 calculated by LAMA algorithm31. b, Two-color STORM images of the RIP1-RIP3 necrosomes in HA-RIP3 expressing HeLa cells treated with TSZ, and labeled for RIP1 and RIP3 (top, “both”), only for RIP3 (middle, “anti-HA only”) or only for RIP1 (bottom, “anti-RIP1 only”). Green indicates mouse secondary antibody conjugated CF 568, and purple indicates rabbit secondary antibody conjugated CF 647. Data are representative of four independent experiments. c, Cross-talk percentages between the two STORM color-channels determined from single-color-labeled necrosome shown in b. Data are mean ± SD of biological triplicates. d,e, Analysis workflow of two-color STORM images in this study (d). Clusters identified by ilastik in each channel were further assessed the possibility of co-clustering in a single channel or overlap between two channels. An example of cluster analysis of two-color STORM imaging is shown in e. The details of segmentation/overlap analysis of clusters are described in the Methods section. Scale bars, 200 nm (e) and 100 nm (b). Statistical source data are provided in Source Data Extended Data Fig. 4.
