Brain Slice Staining and Preparation for Three-Dimensional Super-Resolution Microscopy.

Localization microscopy techniques-such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM)-provide the highest precision for single-molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue. We have developed a sample preparation and image acquisition protocol to address these challenges in rat brain slices. The sample preparation combined multiple fixation steps, saponin permeabilization, and tissue clarification. Together, these preserve intracellular structures, promote antibody penetration, reduce background fluorescence and light scattering, and allow acquisition of images deep in a 30 μm thick slice. Image acquisition challenges were resolved by overlaying samples with a permeable agarose pad and custom-built stainless-steel imaging adapter, and sealing the imaging chamber. This approach kept slices flat, immobile, bathed in imaging buffer, and prevented buffer oxidation during imaging. Using this protocol, we consistently obtained single-molecule localizations of synaptic vesicle and active zone proteins in three dimensions within individual synaptic terminals of the striatum in rat brain slices. These techniques may be easily adapted to the preparation and imaging of other tissues, substantially broadening the application of super-resolution imaging.