Single-molecule, localization-based, super resolution microscopy is able to reveal detailed subcellular structures and protein distributions below the classical ∼250-nm diffraction limit of light, but utilizing this technique effectively requires a combination of careful sample preparation, data acquisition, and data analysis, which can be daunting to novice researchers. In this protocol, detailed instructions on preparation of robust reference samples for super-resolution microscopy, including the cytoskeleton (microtubules), membrane-bound organelles (peroxisomes), and scaffold proteins (clathrin), are provided. These samples also constitute a representative subset of imaging targets of interest to biological researchers and highlight the differences and similarities in sample preparation. © 2015 by John Wiley & Sons, Inc. Copyright © 2013 John Wiley & Sons, Inc. All rights reserved.