Permeabilization-free en bloc immunohistochemistry for correlative microscopy
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) lo...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2021-05-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/63392 |
| _version_ | 1818018743656120320 |
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| author | Kara A Fulton Kevin L Briggman |
| author_facet | Kara A Fulton Kevin L Briggman |
| author_sort | Kara A Fulton |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-14T07:43:53Z |
| format | Article |
| id | doaj.art-ff6cadc4394c4ed78b5e53f58b064555 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-14T07:43:53Z |
| publishDate | 2021-05-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-ff6cadc4394c4ed78b5e53f58b0645552022-12-22T02:05:25ZengeLife Sciences Publications LtdeLife2050-084X2021-05-011010.7554/eLife.63392Permeabilization-free en bloc immunohistochemistry for correlative microscopyKara A Fulton0https://orcid.org/0000-0002-9987-2815Kevin L Briggman1https://orcid.org/0000-0003-2946-4661Brown University, Providence, United States; National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, United States; Center of Advanced European Studies and Research (caesar), Bonn, GermanyNational Institute of Neurological Disorders and Stroke (NINDS), Bethesda, United States; Center of Advanced European Studies and Research (caesar), Bonn, GermanyA 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.https://elifesciences.org/articles/63392Immunohistochemistrycorrelative microscopyelectron microscopyconnectomics |
| spellingShingle | Kara A Fulton Kevin L Briggman Permeabilization-free en bloc immunohistochemistry for correlative microscopy eLife Immunohistochemistry correlative microscopy electron microscopy connectomics |
| title | Permeabilization-free en bloc immunohistochemistry for correlative microscopy |
| title_full | Permeabilization-free en bloc immunohistochemistry for correlative microscopy |
| title_fullStr | Permeabilization-free en bloc immunohistochemistry for correlative microscopy |
| title_full_unstemmed | Permeabilization-free en bloc immunohistochemistry for correlative microscopy |
| title_short | Permeabilization-free en bloc immunohistochemistry for correlative microscopy |
| title_sort | permeabilization free en bloc immunohistochemistry for correlative microscopy |
| topic | Immunohistochemistry correlative microscopy electron microscopy connectomics |
| url | https://elifesciences.org/articles/63392 |
| work_keys_str_mv | AT karaafulton permeabilizationfreeenblocimmunohistochemistryforcorrelativemicroscopy AT kevinlbriggman permeabilizationfreeenblocimmunohistochemistryforcorrelativemicroscopy |