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Permeabilization-free en bloc immunohistochemistry for correlative microscopy.

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.

SUBMITTER: Fulton KA

PROVIDER: S-EPMC8118656 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Permeabilization-free en bloc immunohistochemistry for correlative microscopy.

Fulton Kara A KA Briggman Kevin L KL

eLife 20210513

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 ...[more]

PMID: 33983117

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