Name: toluidine blue staining of resin-embedded sections for evaluation of peripheral nerve morphology
Uploaded: 2018-07-03T15:00:00
Description: Watch this Scientific Journal Video about Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology at JoVE.com

The authors would like to thankthe Jenkins Microscopy Facility at the University of Wyoming for their help, and the members of the Bushman lab, Kelly Roballo, Hayden True, Wupu Osimanjiang and Subash Dhunghana, for assistance in animal care. This publication was made possible by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 2P20GM103432.

Adult Sprague Dawley rats were used in this project and all procedures were approved by the University of Wyoming Institutional Animal Care and Use Committee.
1. Surgery and In Vivo Nerve Fixation
NOTE: Vendor information for all materials and equipment used in this protocol are listed in the Table of Materials.
NOTE: In vivo nerve fixation is used to preserve the tissue and reduce structural degradation t...

<ol>
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Examinations of the morphological structures of peripheral nerve injury and regeneration are frequent subjects of study13. In this protocol, we describe the steps to obtain high quality images for histological data quantifications using rat sciatic nerve tissue embedded in resin blocks and stained with toluidine blue. This technique provides an image of nerve morphology in which the nerve regeneration can be quantified by measuring the number of axons, degree of myelination, presence of infiltrati...

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons1. Peripheral nerve defects caused by medical disorders and trauma represent a major public health concern and have large economic impacts2,3. Despite the advances in assessing the outcomes of peripheral nerve injuries and understanding nerve regeneration, traditional methods such as nerve histology and staining techniques are essential tools to qualitatively and quantitatively assess nerve health as a terminal outcome measurement in animal models or excised human tissue. This is often paired with electrophysiological measurements of peripheral nerve function, where morphometry can reveal why functional nerve regeneration did or did not occur.
Toluidine blue staining of resin embedded semi-thin peripheral nerve sections is a specialized method for imaging myelinated nerve fibers, providing high quality and clear detailed images of nerve structures4,5,6. Toluidine blue is an acidophilic metachromatic stain, discovered by William Henry Perkin in 18567, and has been used in several medical applications8. Toluidine blue-stained peripheral nerve sections obtained from resin-embedded nerve segments allows for clear visualization of nerve structures. Visualization of myelin sheath structure can be enhanced by the use of osmium tetroxide post-fixation4,9. Osmium tetroxide is a toxic oxidant and lipid fixative agent that interacts with the double bonds in lipids, resulting in starkly defined lipid-rich myelin sheaths10. However, osmium tetroxide is toxic, expensive, requires a longer incubation of nerve segments, and is not always used.
Alternative methods of processing and staining have been developed for visualization of peripheral nerve morphology; Paraffin, cryogenic sectioning, and epoxy resin-embedded nerve sectioning followed by staining with toluidine blue or phenylenediamine solution has been used to quantify morphological changes of peripheral nerve regeneration 11,12. These methods each have their advantages and yield essential data on the number of axons, myelin thickness, axon diameter, and axon diameter to myelinated fiber diameter (g-ratio) 11,13,14,15.
The primary distinction of the resin-embedding in this protocol is that it facilitates obtaining 1-2 &#956;m thickness cross-sections due to the hardness of the resin while maintaining the histological qualities of the nerve. These thin sections, as opposed to the 4-5 &#956;m thickness sections obtained from paraffin embedding, provide peripheral nerve sections with higher resolution, allowing for a more accurate quantification of axon myelination, such as the g-ratio, that cannot be obtained from thicker sections16. While cryogenic sectioning can be used to obtain 1-2 &#181;m sections, it has been our experience that it is more difficult to obtain sections without numerous large cracks. Such cracked sections can cause inaccurate counting of the number of axons and aspects of myelination.
In addition to toluidine blue staining17, a silver staining method18 and Masson&#39;s trichrome staining4 can also be used to show nerve axons. However, using resin embedding of rat median nerve sections stained with either hematoxylin and eosin or Masson&#39;s trichrome showed faint myelin sheaths and unrecognized structures, whereas toluidine blue staining showed clear myelin sheath image and easily can be quantified4. Despite some limitations, toluidine blue staining of resin embedded peripheral nerves is a valuable technique that can be used when high resolution images of nerve morphology are required.
The primary disadvantage for resin embedding is that it is time-consuming and does not allow for immunostaining of the same tissue due to the difficulty of antigen retrieval when compared to paraffin and frozen embedded sections techniques. Thus, it is not generally possible to utilize the same tissue for immunostaining that is processed via resin-embedding for toluidine blue staining. Although not used here, if immunohistochemistry is desired in resin embedded sections, the use of glycol methacrylate embedding resins allows for immunohistochemistry to be performed on tissue sections, but it is relatively expensive19. This can be somewhat mitigated by cutting the peripheral nerve into separate segments, some for resin-embedding and others for immunostaining directly after fixation.
The process of toluidine blue staining of resin embedded peripheral nerves, as with most histopathological analysis, can be broken up into five stages, including fixation, dehydration, embedding, sectioning, and staining20. We aim here to provide a protocol and practical guideline for using resin embedded rat sciatic nerve sections stained with toluidine blue to acquire high quality images.

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toluidine blue staining of resin-embedded sections for evaluation of peripheral nerve morphology

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves are frequently damaged because of trauma or disease. As methods and strategies have been developed to assess peripheral nerve injury in animal models, function and regeneration, analyzing the morphometry of the peripheral nerve has become an essential terminal outcome measurement. Toluidine blue staining of nerve cross sections obtained from resin embedded nerve sections is a reproducible method for qualitative and quantitative assessments of peripheral nerves, enabling visualization of morphology number of axons and degree of myelination. This technique, as with many other histological methods, can be difficult to learn and master using standard written protocols. The intent of this publication is therefore to accentuate written protocols for toluidine blue staining of peripheral nerves with videography of the method, using sciatic nerves harvested from rats. In this protocol, we describe in vivo peripheral nerve fixation and collection of the tissue, and post-fixation with 2% osmium tetroxide, embedding of nerves in epoxy resin, and ultramicrotome sectioning of nerves to 1-2&#956;m thickness. Nerve sections then transferred to a glass slide and stained with toluidine blue, after which they are quantitatively and qualitatively assessed. Examples of the most common problems are shown, as well as steps for mitigating these issues.

Resin embedded peripheral nerve sections stained with toluidine blue allow for accurate histological data quantifications. An overview of the procedure is shown in (Figure 2). Sciatic nerves sections embedded in resin medium and stained with toluidine blue showed clear images with optimal resolution (Figures 3). Nerve damage can cause many changes in nerve morphological structures, for example, changes in nerve fiber, axon diameter, and myeli...

Watch this Scientific Journal Video about Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology at JoVE.com

Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology

Here we present a protocol to visualize fine structures of peripheral nerves by obtaining and staining 1-2 &#181;m sections with toluidine blue

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves ...

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Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

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 ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...