Single-molecule localization microscopy (SMLM) is rapidly gaining popularity in the
life sciences as an efficient approach to visualize molecular distribution with nanoscale
precision. However, it has been challenging to obtain and analyze such data within
a cellular context in tissue preparations. Here we describe a 5-d tissue processing
and immunostaining procedure that is optimized for SMLM, and we provide example applications
to fixed mouse brain, heart and kidney tissues. We then describe how to perform correlated
confocal and 3D-superresolution imaging on these sections, which allows the visualization
of nanoscale protein localization within labeled subcellular compartments of identified
target cells in a few minutes. Finally, we describe the use of VividSTORM (http://katonalab.hu/index.php/vividstorm),
an open-source software for correlated confocal and SMLM image analysis, which facilitates
the measurement of molecular abundance, clustering, internalization, surface density
and intermolecular distances in a cell-specific and subcellular compartment-restricted
manner. The protocol requires only basic skills in tissue staining and microscopy.
