Fig. 1: Unsuccessful and successful staining for 3-color SMLM in multiple cell lines.
From: Three-color single-molecule localization microscopy in chromatin

a Schematic showing the difficulties of multi-labeling in a dense nuclear environment relative to sparse cytoplasm. The first panel showcases microtubules being labeled by antibody complex (primary and secondary are shown as one circle for clarity in the diagram). In a sparse environment, the antibodies are not hindered by neighboring structures and can reach their target. In the middle panel, we showcase the difficulty of getting antibodies in the nucleus, with some reaching the interior and labeling chromatin, while the majority lies outside the nuclear envelope. The third panel illustrates the issues of labeling a target within the dense nucleus: cross-reactivity shown by Antibody Complex I (which in this example aims to label RNAPII) reacting with other nuclear macromolecules, cross talk due to proximity, and adsorption to non-target surfaces due to high crowding. We have added red arrows to showcase these examples within the diagram. b Top row shows the failing staining of H3K9me3, H3K27ac, RNA Polymerase II in OVCAR5 cells, based on simultaneous staining protocol, and their merge image respectfully, while b bottom row displays the successful staining by sequential staining protocol in HeLa cells. All images scale bar is 5 µm. c Exemplary three-label SMLM images targeting H3K9me3, H3K27ac, and RNAPII for BJ Fibroblast, HCT116, and MCF10A cell lines. All scale bars are 3 µm