Looking through Brains with Fast Passive CLARITY: Zebrafish, Rodents, Non-human Primates and Humans

Mortazavi, Farzad; Stankiewicz, Alexander J.; Zhdanova, Irina V.

doi:10.21769/bioprotoc.3321

Cited by 6 publications

(5 citation statements)

References 61 publications

(12 reference statements)

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“…Concerning the aqueous hyperhydrating-based tissue-clearing methods, our results showed that FJ-C is compatible with SeeDB2 and CUBIC-L but not RTF and CUBIC-R. Since the purpose of CUBIC-R incubation is to homogenize the refractive index of the tissue, and is not directly involved in tissue clearing per se, CUBIC-R incubation may be omitted or substituted by another incubation 33 and the pClarity method can cause tissue expansion, 32 the labeled cells overall in (A) appear smaller than those in (B). medium that could homogenize refractive index and is compatible with FJ-C.…”

Section: Discussionmentioning

confidence: 85%

“…Scale bar indicates 50 µm for both panels. Because the RTF method can cause tissue shrinkage 33 and the pClarity method can cause tissue expansion, 32 the labeled cells overall in (A) appear smaller than those in (B).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Tissue samples were incubated in a borate-SDS (sodium dodecyl sulfate) buffer containing 200 mM boric acid, 4% SDS, and 20 mM lithium hydroxide monohydrate (pH 8.5) at 37°C until tissue became transparent. 32 RTF (Rapid clearing method based on Triethanolamine and Formamide). Tissue samples were incubated in RTF-R1 (30% triethanolamine, 40% formamide, and 30% dH 2 O) for 2-3 h, in RTF-R2 (60% triethanolamine, 25% formamide, and 15% dH 2 O) for 2-3 h, and in RTF-R3 (70% triethanolamine, 15% formamide and 15% dH 2 O) for at least one day.…”

Section: Cubic (Clear Unobstructed Brain/body Imaging Cocktails)mentioning

confidence: 99%

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Combining tissue clearing and Fluoro-Jade C labeling for neurotoxicity assessments

Sarkar

Raymick

et al. 2023

Exp Biol Med (Maywood)

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Tissue clearing refers to laboratory methods that make tissue transparent by chemical means. This approach allows the labeling, visualization, and analysis of specific targets without cutting the tissue into sections, thereby maintaining three-dimensional architecture. More than two dozen tissue-clearing methods have been developed by different research teams to date. While tissue clearing has been successfully applied in several studies concerning basic science or diseases, little is known about the utilization of tissue clearing for neurotoxicity evaluation. In this study, several tissue-clearing methods were combined with Fluoro-Jade C (FJ-C), a standard marker of neurodegeneration. The results suggest that some but not all tissue-clearing media are compatible with the FJ-C fluorophore. By utilizing a neurotoxicity animal model, the results further suggest that FJ-C labeling can be combined with tissue clearing for neurotoxicity assessments. This approach has the potential to be expanded further by combining multicolor labeling of molecular targets involved in the development and/or mechanisms of neurotoxicity and neurodegeneration.

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“…The major component of passive tissue clearing is still SDS but the tissue is subjected to hydrogel polymerization prior to the SDS solution. This causes the increased swelling but it is better suited for applications where preserving RNA and/or DNA is needed (Mortazavi, Stankiewicz, & Zhdanova, 2019).…”

Section: Clearing Brain Tissue With Passive Claritymentioning

confidence: 99%

Characterization of Astrocyte Morphology and Function Using a Fast and Reliable Tissue Clearing Technique

et al. 2021

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Astrocytic processes interact with synapses throughout the brain modulating neurotransmitter signaling and synaptic communication. During conditions such as exposure to drugs of abuse and neurological diseases, astrocytes respond by altering their morphological and functional properties. Reactive astrocyte phenotypes exhibit a bushy morphology with altered soma volume and an increased number of processes compared to resting astrocytes. The reactive astrocytic phenotype also overexpresses proteins one of which can be glial fibrillary acidic protein (GFAP). Fluorescence microscopy on thin tissue sections (<20 μm) requires reconstruction, often through multiple sections, to delineate the full astrocytic morphology. In contrast, tissue clearing methods have been developed that enable imaging of larger sections including the whole brain, providing an opportunity to see in-depth changes in single cell structure. In this article, a detailed protocol for studying astrocyte morphology using tissue clearing and subsequent imaging of whole brains as well as region-specific slices is provided. This method is ideal for understanding the effect of different physiological conditions on astrocyte morphology. A standard biochemistry laboratory has the resources to accomplish tissue clearing using this protocol and most universities have the required imaging facilities. Protocols to study brains from both genetically modified mice that contain an astrocyte-specific marker and from wild-type mice using antibody labeling steps after tissue clearing are provided. We also describe general protocols to conduct fluorescence imaging of astrocytes in cleared tissue to characterize their morphology. This protocol could be useful for researchers working in the rapidly growing field of astrocyte biology.

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Looking through Brains with Fast Passive CLARITY: Zebrafish, Rodents, Non-human Primates and Humans

Cited by 6 publications

References 61 publications

Combining tissue clearing and Fluoro-Jade C labeling for neurotoxicity assessments

Characterization of Astrocyte Morphology and Function Using a Fast and Reliable Tissue Clearing Technique

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