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.
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(a) Hippocampal GABAergic interneurons were filled with biocytin during whole-cell patch-clamp electrophysiological recording in acute slice preparation, and then the biocytin labeling was developed with Alexa Fluor 488–streptavidin according to PROCEDURE Steps 1–8. The maximal intensity projection of the deconvolved confocal image shows the axon of the filled neuron with an array of axon terminals appearing as varicosities. (b) Immunostaining against the CB1 cannabinoid receptor was visualized using a secondary antibody coupled with Alexa Fluor 405–Alexa Fluor 647 activator-reporter dye pairs, and imaged in normal STORM mode according to PROCEDURE Steps 9–33. (c) The corresponding confocal and rendered STORM images from the sample are overlaid, identifying the localization points that belong to the biocytin-filled axon. (d,e) The enlarged view of the STORM (d) and overlay (e) images from the boxed region in a demonstrates high CB1 receptor density on the target axon terminal. The experiment was approved by the Hungarian Committee of the Scientific Ethics of Animal Research.