An optimized tissue clearing protocol for rat brain labeling, imaging, and high throughput analysis

Branch, Audrey; Tward, Daniel J.; Kolstad, Anna C; Pulyadi, Vyash; Vogelstein, Joshua T.; Wu, Zhuhao; Gallagher, Michela

doi:10.1101/639674

Cited by 8 publications

(4 citation statements)

References 20 publications

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“…This may be due to its polarizable sulfur atom (see Table 2 ). Additionally, iDISCO+ has been combined with Adipo-Clear (see section Adipose Tissue) to improve the clearing of the whole rat brain by further optimization of the delipidation step ( Branch et al, 2019 ).…”

Section: Translational Applications Of Tissue Clearing and 3d Imagingmentioning

confidence: 99%

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

et al. 2020

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Summary Three-dimensional (3D) optical imaging techniques can expand our knowledge about physiological and pathological processes that cannot be fully understood with 2D approaches. Standard diagnostic tests frequently are not sufficient to unequivocally determine the presence of a pathological condition. Whole-organ optical imaging requires tissue transparency, which can be achieved by using tissue clearing procedures enabling deeper image acquisition and therefore making possible the analysis of large-scale biological tissue samples. Here, we review currently available clearing agents, methods, and their application in imaging of physiological or pathological conditions in different animal and human organs. We also compare different optical tissue clearing methods discussing their advantages and disadvantages and review the use of different 3D imaging techniques for the visualization and image acquisition of cleared tissues. The use of optical tissue clearing resources for large-scale biological tissues 3D imaging paves the way for future applications in translational and clinical research.

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Section: Translational Applications Of Tissue Clearing and 3d Imagingmentioning

confidence: 99%

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

et al. 2020

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“…We addressed this in two ways. First, we implemented a modified version of the AdipoClear tissue clearing protocol (34,35) that reduces the autofluorescence of myelin. As fiber tracts composed of unlabeled axons share characteristics with the labeled axons we aim to extract, this reduces the rate of false positives in structures such as the striatum (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Mapping mesoscale axonal projections in the mouse brain using a 3D convolutional network

Friedmann

Pun

Adams

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The projection targets of a neuronal population are a key feature of its anatomical characteristics. Historically, tissue sectioning, confocal microscopy, and manual scoring of specific regions of interest have been used to generate coarse summaries of mesoscale projectomes. We present here TrailMap, a three-dimensional (3D) convolutional network for extracting axonal projections from intact cleared mouse brains imaged by light-sheet microscopy. TrailMap allows region-based quantification of total axon content in large and complex 3D structures after registration to a standard reference atlas. The identification of axonal structures as thin as one voxel benefits from data augmentation but also requires a loss function that tolerates errors in annotation. A network trained with volumes of serotonergic axons in all major brain regions can be generalized to map and quantify axons from thalamocortical, deep cerebellar, and cortical projection neurons, validating transfer learning as a tool to adapt the model to novel categories of axonal morphology. Speed of training, ease of use, and accuracy improve over existing tools without a need for specialized computing hardware. Given the recent emphasis on genetically and functionally defining cell types in neural circuit analysis, TrailMap will facilitate automated extraction and quantification of axons from these specific cell types at the scale of the entire mouse brain, an essential component of deciphering their connectivity.

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“…Tissue permeabilization is the first concern. Alternative detergents e.g., CHAPS (Frankowski et al, 2022 ) and β-cyclodextrin (Branch et al, 2021 ; Mai et al, 2023 ) drastically facilitate deeper immunostaining. Whether each antibody is compatible with a new chemical has to be carefully tested with brain slice IHC.…”

Section: Tissue Clearing For Brain-wide Engram Mappingmentioning

confidence: 99%

Tissue clearing applications in memory engram research

Yip,

Gräff

2023

Front. Behav. Neurosci.

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A memory engram is thought to be the physical substrate of the memory trace within the brain, which is generally depicted as a neuronal ensemble activated by learning to fire together during encoding and retrieval. It has been postulated that engram cell ensembles are functionally interconnected across multiple brain regions to store a single memory as an “engram complex”, but visualizing this engram complex across the whole brain has for long been hindered by technical limitations. With the recent development of tissue clearing techniques, advanced light-sheet microscopy, and automated 3D image analysis, it has now become possible to generate a brain-wide map of engram cells and thereby to visualize the “engram complex”. In this review, we first provide a comprehensive summary of brain-wide engram mapping studies to date. We then compile a guide on implementing the optimal tissue clearing technique for engram tagging approaches, paying particular attention to visualize engram reactivation as a critical mnemonic property, for which whole-brain multiplexed immunostaining becomes a challenging prerequisite. Finally, we highlight the potential of tissue clearing to simultaneously shed light on both the circuit connectivity and molecular underpinnings of engram cells in a single snapshot. In doing so, novel brain regions and circuits can be identified for subsequent functional manipulation, thus providing an opportunity to robustly examine the “engram complex” underlying memory storage.

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An optimized tissue clearing protocol for rat brain labeling, imaging, and high throughput analysis

Cited by 8 publications

References 20 publications

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

Mapping mesoscale axonal projections in the mouse brain using a 3D convolutional network

Tissue clearing applications in memory engram research

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