Combining Dstorm With Proximity Ligation Assays For Multi-Color Colocalization

Many cellular processes are controlled by the stable or transient association of proteins. Fine details of the colocalization of small molecules can be observed in cells by two-color super-resolved localization microscopy techniques. Alternatively, in situ proximity ligation assay (in situ PLA) is an immunolabeling strategy used with conventional microscopy for colocalizing proteins in situ, but with only a single dye.We directly compare colocalization results obtained through two-color direct stochastic optical reconstruction microscopy (dSTORM) with in situ PLA by imaging immunolabeled RNAPII in the nucleus of mouse 3T3 fibroblast cells. Two domains of the RNAPII are targets for the antibodies: the carboxyl terminal domain (CTD) and the core domain. During transcription, the carboxyl terminal domain (CTD) of RNA Polymerase II (RNAPII) becomes phosphorylated and those phosphorylated sites can be targeted by antibodies. Therefore, only active RNAPII should contain both antibodies, while a soluble pool of RNAPII should only contain a label on the core, allowing us to differentiate between these populations.Finally, we present future work in combining these two methods to study the interaction of multiple-proteins with a limited number of dyes.