This document is intended as a quick reference guide for the most common questions regarding the preparation of samples for the Leica SP8 TCS STED
3X, by briefly explaining the theoretical basics required from samples for stimulated emission depletion. It provides information about the most common immuno-fluorescence labeling techniques, working mounting media, basic quality optimization procedures and experiment designs. It also contains a detailed list of reagents, antibodies and disposables frequently and successfully used in super-resolution STED
microscopy. In summary, this guide is meant to present the necessary knowledge, including some tips and tricks, to users that are preparing their first super-resolution microscopy sample.