The preparation of samples and the choice of appropriate labeling techniques have become instrumental for the development of light microscopy techniques with increasingly high resolution. Both localization microscopy and STED approaches require fluorophores with specific features, including high photostability, specific excitation–emission spectra, and selective switching of single molecules to “on” and “off” state. Additionally, at higher resolutions the limits of conventional immunostaining often become apparent, as clearly exemplified by rather fragmented stainings of continuous cellular components such as microtubules and membranous organelles. Hence, the correct exploitation of fluorescent probes is of crucial importance for successful super-resolution imaging. Here, the most prominent techniques related to super-resolution imaging are briefly explained, followed by a more detailed technical description of fluorophores and embedding media that are required for such imaging procedures. Some relevant aspects of troubleshooting the preparation of super-resolution samples are included to offer practical support for such experiment.