Permeabilization-free en bloc immunohistochemistry for correlative microscopy

eLife

Kara A Fulton¹  Kevin L Briggman² 

[1] Brown University, Providence, United States;National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, United States;Center for the Advancement of European Science and Research (caesar), Bonn, Germany;National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, United States;Center for the Advancement of European Science and Research (caesar), Bonn, Germany;

关键词: Immunohistochemistry;    correlative microscopy;    electron microscopy;    connectomics;    Mouse;   

DOI  :  10.7554/eLife.63392

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY