Abstract
A dense reconstruction of neuronal synaptic connectivity typically requires high-resolution 3D electron microscopy (EM) data, but EM data alone lacks functional information about neurons and synapses. One approach to augment structural EM datasets is with the fluorescent immunohistochemical (IHC) localization of functionally relevant proteins. We describe a protocol that obviates the requirement of tissue permeabilization in thick tissue sections, a major impediment for correlative pre-embedding IHC and EM. We demonstrate the permeabilization-free labeling of neuronal cell types, intracellular enzymes, and synaptic proteins in tissue sections hundreds of microns thick in multiple brain regions from mice while simultaneously retaining the ultrastructural integrity of the tissue. Finally, we explore the utility of this protocol by performing proof-of-principle correlative experiments combining two-photon imaging of protein distributions and 3D EM.
Highlights
Anatomical reconstructions of synaptic connectivity are one essential component of understanding neuronal circuits in the nervous system
When detergent was omitted from the labeling protocol, we observed a gradient of fluorescently labeled somata that decreased in the center of sections, as expected, and the ultrastructural membrane integrity of the tissue remained intact (Figure 1b)
When detergent was omitted from the protocol, the labeling of neuronal somata in extracellular space (ECS)-preserved sections remained uniform throughout the tissue (Figure 1d)
Summary
Anatomical reconstructions of synaptic connectivity are one essential component of understanding neuronal circuits in the nervous system. Array tomography was developed to multiplex the labeling of numerous targets in ultrathin tissue sections (Collman et al, 2015; Micheva and Smith, 2007). This approach is incompatible with en bloc-based methods for collecting 3D EM data that require pre-embedding IHC, as in the case of focused ion beam scanning electron microscopy (FIB-SEM, Hayworth et al, 2015; Knott et al, 2008), gas cluster ion beam SEM (Hayworth et al, 2020), and serial block-face scanning electron microscopy (SBEM, Denk and Horstmann, 2004). For pre-embedding IHC, a membrane permeabilization step with nonionic surfactants such as Triton X-100 or polysorbate 20 (Tween 20) is typically performed to enable antibody penetration into thick tissue blocks, but at the cost of degraded tissue ultrastructure (Helenius and Simons, 1975; Humbel et al, 1998)
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