SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue
Postmortem human brain tissue is a critical resource for studying neurodegenerative disease, providing critical insights into cellular morphology, pathology, and network connectivity. To improve standard microscopy and enable high-resolution, three-dimensional (3D) images of tissues at the subcellul...
Saved in:
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2024-10-01
|
| Series: | Frontiers in Neuroscience |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fnins.2024.1474617/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849411964564930560 |
|---|---|
| author | Grace A. Rosen Grace A. Rosen Grace A. Rosen Daniel Kirsch Raymond Nicks Hunter Kelley Hunter Kelley Hunter Kelley Rebecca Mathias Rebecca Mathias Kerry A. Cormier Kerry A. Cormier Kerry A. Cormier Kerry A. Cormier Caroline A. Kubilus Caroline A. Kubilus Caroline A. Kubilus Bryan Dec Thor D. Stein Thor D. Stein Thor D. Stein Thor D. Stein Victor E. Alvarez Victor E. Alvarez Victor E. Alvarez Victor E. Alvarez Victor E. Alvarez Michael L. Alosco Michael L. Alosco Michael L. Alosco Michael L. Alosco Ann C. McKee Ann C. McKee Ann C. McKee Ann C. McKee Ann C. McKee Bertrand R. Huber Bertrand R. Huber Bertrand R. Huber Bertrand R. Huber |
| author_facet | Grace A. Rosen Grace A. Rosen Grace A. Rosen Daniel Kirsch Raymond Nicks Hunter Kelley Hunter Kelley Hunter Kelley Rebecca Mathias Rebecca Mathias Kerry A. Cormier Kerry A. Cormier Kerry A. Cormier Kerry A. Cormier Caroline A. Kubilus Caroline A. Kubilus Caroline A. Kubilus Bryan Dec Thor D. Stein Thor D. Stein Thor D. Stein Thor D. Stein Victor E. Alvarez Victor E. Alvarez Victor E. Alvarez Victor E. Alvarez Victor E. Alvarez Michael L. Alosco Michael L. Alosco Michael L. Alosco Michael L. Alosco Ann C. McKee Ann C. McKee Ann C. McKee Ann C. McKee Ann C. McKee Bertrand R. Huber Bertrand R. Huber Bertrand R. Huber Bertrand R. Huber |
| author_sort | Grace A. Rosen |
| collection | DOAJ |
| description | Postmortem human brain tissue is a critical resource for studying neurodegenerative disease, providing critical insights into cellular morphology, pathology, and network connectivity. To improve standard microscopy and enable high-resolution, three-dimensional (3D) images of tissues at the subcellular level, tissue-clearing methods have been developed. These 3D images allow for the analysis of large regions of interest and can be used to study structural and spatial changes that occur during neurodegeneration. Additionally, 3D imaging facilitates the visualization of whole-cell morphology, especially in cells with long processes that would otherwise be truncated in single-plane images. Human brain tissue is especially challenging for tissue clearing due to the abundance of lipids in myelin and the need for optimal fixation and low postmortem intervals. Formaldehyde-based fixatives, commonly used in preserving tissue, hinder antibody binding by crosslinking important antibody epitopes, and fluorescent microscopy requires the incorporation of fluorescent labels through passive diffusion or electrophoresis. Recent studies have focused on optimally fixed human brain tissue with short postmortem intervals, limiting the general applicability of these methods. To address these challenges, we developed SHARD (SHIELD, antigen retrieval, and delipidation), a simple and widely applicable method for clearing and labeling human brain tissue, which can be applied to long-term banked human brain tissue preserved in formaldehyde. SHARD is a novel addition to the SHIELD tissue clarification method, combining antigen retrieval, tissue clearing, and staining of 200-μm sections from long-term banked human brain tissue. The SHARD method is effective for postmortem intervals (PMIs) ranging from 10 to 72 h in multiple neurodegenerative diseases and control samples. In this study, we demonstrate that the SHARD method significantly enhances the immunostaining of glial fibrillary acidic protein (GFAP), an astrocytic cytoskeletal marker. Overall, the combination of antigen retrieval and tissue delipidation holds great potential for achieving detailed 3D immunostaining in long-term formaldehyde-fixed postmortem human brain tissue, opening new avenues for research and discovery. |
| format | Article |
| id | doaj-art-5196c8fe3c9443aaa0ad2245589b33e4 |
| institution | Kabale University |
| issn | 1662-453X |
| language | English |
| publishDate | 2024-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Neuroscience |
| spelling | doaj-art-5196c8fe3c9443aaa0ad2245589b33e42025-08-20T03:34:36ZengFrontiers Media S.A.Frontiers in Neuroscience1662-453X2024-10-011810.3389/fnins.2024.14746171474617SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissueGrace A. Rosen0Grace A. Rosen1Grace A. Rosen2Daniel Kirsch3Raymond Nicks4Hunter Kelley5Hunter Kelley6Hunter Kelley7Rebecca Mathias8Rebecca Mathias9Kerry A. Cormier10Kerry A. Cormier11Kerry A. Cormier12Kerry A. Cormier13Caroline A. Kubilus14Caroline A. Kubilus15Caroline A. Kubilus16Bryan Dec17Thor D. Stein18Thor D. Stein19Thor D. Stein20Thor D. Stein21Victor E. Alvarez22Victor E. Alvarez23Victor E. Alvarez24Victor E. Alvarez25Victor E. Alvarez26Michael L. Alosco27Michael L. Alosco28Michael L. Alosco29Michael L. Alosco30Ann C. McKee31Ann C. McKee32Ann C. McKee33Ann C. McKee34Ann C. McKee35Bertrand R. Huber36Bertrand R. Huber37Bertrand R. Huber38Bertrand R. Huber39VA Boston Healthcare System, US Department of Veterans Affairs, Boston, MA, United StatesDepartment of Neurology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesNational Center for PTSD, US Department of Veterans Affairs, Boston, MA, United StatesDepartment of Pathology and Laboratory Medicine, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesBoston University Alzheimer's Disease Research Center and Boston University CTE Center, Boston, MA, United StatesVA Boston Healthcare System, US Department of Veterans Affairs, Boston, MA, United StatesNational Center for PTSD, US Department of Veterans Affairs, Boston, MA, United StatesBoston University Alzheimer's Disease Research Center and Boston University CTE Center, Boston, MA, United StatesVA Boston Healthcare System, US Department of Veterans Affairs, Boston, MA, United StatesDepartment of Neurology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesVA Boston Healthcare System, US Department of Veterans Affairs, Boston, MA, United StatesDepartment of Neurology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesBoston University Alzheimer's Disease Research Center and Boston University CTE Center, Boston, MA, United StatesVA Bedford Healthcare System, US Department of Veterans Affairs, Bedford, MA, United StatesDepartment of Neurology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesBoston University Alzheimer's Disease Research Center and Boston University CTE Center, Boston, MA, United StatesVA Bedford Healthcare System, US Department of Veterans Affairs, Bedford, MA, United StatesBoston University Alzheimer's Disease Research Center and Boston University CTE Center, Boston, MA, United StatesVA Boston Healthcare System, US Department of Veterans Affairs, Boston, MA, United StatesDepartment of Neurology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesDepartment of Pathology and Laboratory Medicine, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesBoston University Alzheimer's Disease Research Center and Boston University CTE Center, Boston, MA, United StatesVA Boston Healthcare System, US Department of Veterans Affairs, Boston, MA, United StatesDepartment of Neurology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesNational Center for PTSD, US Department of Veterans Affairs, Boston, MA, United StatesBoston University Alzheimer's Disease Research Center and Boston University CTE Center, Boston, MA, United StatesVA Bedford Healthcare System, US Department of Veterans Affairs, Bedford, MA, United StatesDepartment of Neurology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesBoston University Alzheimer's Disease Research Center and Boston University CTE Center, Boston, MA, United StatesDepartment of Neurology, Boston Medical Center, Boston, MA, United StatesDepartment of Anatomy and Neurobiology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesVA Boston Healthcare System, US Department of Veterans Affairs, Boston, MA, United StatesDepartment of Neurology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesDepartment of Pathology and Laboratory Medicine, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesBoston University Alzheimer's Disease Research Center and Boston University CTE Center, Boston, MA, United StatesVA Bedford Healthcare System, US Department of Veterans Affairs, Bedford, MA, United StatesVA Boston Healthcare System, US Department of Veterans Affairs, Boston, MA, United StatesDepartment of Neurology, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, United StatesNational Center for PTSD, US Department of Veterans Affairs, Boston, MA, United StatesBoston University Alzheimer's Disease Research Center and Boston University CTE Center, Boston, MA, United StatesPostmortem human brain tissue is a critical resource for studying neurodegenerative disease, providing critical insights into cellular morphology, pathology, and network connectivity. To improve standard microscopy and enable high-resolution, three-dimensional (3D) images of tissues at the subcellular level, tissue-clearing methods have been developed. These 3D images allow for the analysis of large regions of interest and can be used to study structural and spatial changes that occur during neurodegeneration. Additionally, 3D imaging facilitates the visualization of whole-cell morphology, especially in cells with long processes that would otherwise be truncated in single-plane images. Human brain tissue is especially challenging for tissue clearing due to the abundance of lipids in myelin and the need for optimal fixation and low postmortem intervals. Formaldehyde-based fixatives, commonly used in preserving tissue, hinder antibody binding by crosslinking important antibody epitopes, and fluorescent microscopy requires the incorporation of fluorescent labels through passive diffusion or electrophoresis. Recent studies have focused on optimally fixed human brain tissue with short postmortem intervals, limiting the general applicability of these methods. To address these challenges, we developed SHARD (SHIELD, antigen retrieval, and delipidation), a simple and widely applicable method for clearing and labeling human brain tissue, which can be applied to long-term banked human brain tissue preserved in formaldehyde. SHARD is a novel addition to the SHIELD tissue clarification method, combining antigen retrieval, tissue clearing, and staining of 200-μm sections from long-term banked human brain tissue. The SHARD method is effective for postmortem intervals (PMIs) ranging from 10 to 72 h in multiple neurodegenerative diseases and control samples. In this study, we demonstrate that the SHARD method significantly enhances the immunostaining of glial fibrillary acidic protein (GFAP), an astrocytic cytoskeletal marker. Overall, the combination of antigen retrieval and tissue delipidation holds great potential for achieving detailed 3D immunostaining in long-term formaldehyde-fixed postmortem human brain tissue, opening new avenues for research and discovery.https://www.frontiersin.org/articles/10.3389/fnins.2024.1474617/fulloptical clearingantigen retrievalvolumetric imaging3D confocal imaging3D confocal laser scanning microscopyhuman brain banking |
| spellingShingle | Grace A. Rosen Grace A. Rosen Grace A. Rosen Daniel Kirsch Raymond Nicks Hunter Kelley Hunter Kelley Hunter Kelley Rebecca Mathias Rebecca Mathias Kerry A. Cormier Kerry A. Cormier Kerry A. Cormier Kerry A. Cormier Caroline A. Kubilus Caroline A. Kubilus Caroline A. Kubilus Bryan Dec Thor D. Stein Thor D. Stein Thor D. Stein Thor D. Stein Victor E. Alvarez Victor E. Alvarez Victor E. Alvarez Victor E. Alvarez Victor E. Alvarez Michael L. Alosco Michael L. Alosco Michael L. Alosco Michael L. Alosco Ann C. McKee Ann C. McKee Ann C. McKee Ann C. McKee Ann C. McKee Bertrand R. Huber Bertrand R. Huber Bertrand R. Huber Bertrand R. Huber SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue Frontiers in Neuroscience optical clearing antigen retrieval volumetric imaging 3D confocal imaging 3D confocal laser scanning microscopy human brain banking |
| title | SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue |
| title_full | SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue |
| title_fullStr | SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue |
| title_full_unstemmed | SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue |
| title_short | SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue |
| title_sort | shard an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue |
| topic | optical clearing antigen retrieval volumetric imaging 3D confocal imaging 3D confocal laser scanning microscopy human brain banking |
| url | https://www.frontiersin.org/articles/10.3389/fnins.2024.1474617/full |
| work_keys_str_mv | AT gracearosen shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT gracearosen shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT gracearosen shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT danielkirsch shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT raymondnicks shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT hunterkelley shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT hunterkelley shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT hunterkelley shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT rebeccamathias shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT rebeccamathias shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT kerryacormier shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT kerryacormier shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT kerryacormier shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT kerryacormier shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT carolineakubilus shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT carolineakubilus shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT carolineakubilus shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT bryandec shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT thordstein shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT thordstein shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT thordstein shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT thordstein shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT victorealvarez shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT victorealvarez shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT victorealvarez shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT victorealvarez shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT victorealvarez shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT michaellalosco shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT michaellalosco shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT michaellalosco shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT michaellalosco shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT anncmckee shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT anncmckee shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT anncmckee shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT anncmckee shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT anncmckee shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT bertrandrhuber shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT bertrandrhuber shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT bertrandrhuber shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue AT bertrandrhuber shardanimprovedmethodforstainingandvisualizingmultipleximmunofluorescenceinopticallyclearedpostmortemhumanbraintissue |