Name: Toluidine Blue Staining of Peripheral Nerve Sections: A Technique to Stain Myelinated Nerve Fibers For Peripheral Nerve Structure Visualization
Uploaded: 2023-04-30T14:58:04
Description: Source: Ghnenis, A. B. et al., Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology.&#160;J. Vis. ...

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1. Toluidine Blue Staining
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	<li>Prepare 1% solution of toluidine blue by dissolving 2 g of sodium borate in 100 mL of deionized water, then add 1 g of toluidine blue and stir until dissolved. Filter the solution using filter paper (pore size: 11 &#181;m) and keep the solution in an opaque bottle at room temperature for up to tw...

toluidine blue staining of peripheral nerve sections: a technique to stain myelinated nerve fibers for peripheral nerve structure visualization

Source: Ghnenis, A. B. et al., <a href="https://www.jove.com/v/58031">Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology.</a>&#160;J. Vis. Exp.&#160;(2018)
This video demonstrates the staining technique of resin-embedded, osmium tetroxide-treated rat peripheral nerve sections using toluidine blue. The stained nerve sections help visualize the fine structures of peripheral nerves.

Toluidine Blue Staining of Peripheral Nerve Sections: A Technique to Stain Myelinated Nerve Fibers For Peripheral Nerve Structure Visualization

Source: Ghnenis, A. B. et al., Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology.&#160;J. Vis. ...

Peripheral nerves consist of axons surrounded by Schwann cells that wrap around the axons with multiple layers of lipid-rich cell membranes, forming the myelin sheath.
To visualize peripheral nerves, begin with a glass slide carrying a fixed, resin-embedded, osmium tetroxide-treated transverse rat peripheral nerve section. Resin-embedded sections help maintain tissue morphology.
Osmium tetroxide treatment causes deposition of metallic osmium in the lipid layer of myelin sheath at the tissue site.
Pipette alkaline solution of toluidine blue over the tissue section. Dry the slide at an appropriate temperature. The alkaline conditions and heat facilitate toluidine blue penetration through the resin into the tissue section.
Toluidine blue, being a cationic dye, binds to the negatively-charged phosphate groups on DNA and also intercalates between the DNA base pairs in Schwann cell nuclei. In addition, the dye binds to RNA in the cytoplasm, outside the myelin sheath.
Add a mounting solution over the stained nerve section and cover with a coverslip to prepare the tissue for imaging. Under a light microscope, myelin sheath layers appear black with defined myelinated nerve fiber structures following osmium treatment, while the axons remain unstained.&#160;
Schwann cell nuclei and cytoplasm appear different shades of blue due to metachromatic property of the dye, facilitating visualization of axon morphology and degree of myelination in peripheral nerves.

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Tissue staining

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

JoVE Journal

Immunology and Infection

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

Neuroscience

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

Toluidine Blue Staining of Peripheral Nerve Sections: A Technique to Stain Myelinated Nerve Fibers For Peripheral Nerve Structure Visualization

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