Name: tissue preparation and immunostaining of mouse sensory nerve fibers innervating skin and limb bones
Uploaded: 2012-01-26T12:00:00
Description: Watch this Scientific Journal Video about Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones at JoVE.com

We thank Dr. Yuriy M. Usachev for his help in the initial standardization of confocal microscopy/imaging of mouse plantar punch biopsy immunostaining; and Dr. Donna L. Hammond for her continued help and constructive criticism in this work. This work was funded by grants from the NINDS/NIH (NS069898), and an Idea Development Grant Award from the Department of Defense Prostate Cancer Research Program (DoD-PCRP-101096) to D.P.M.

1. Animal Perfusion
All animal procedures performed in this study are approved by the Institutional Animal Care and Use Committee of the University of Iowa, and follow NIH guidelines for the use of animals in research.
<ol>
	<li>On the day before perfusion, prepare 1 L of phosphate buffer (0.2 M PB in double distilled H2O, pH 7.4), and store at 4&deg;C. This will be used for perfusion and post-fixation processes.</li>
	<li>On the day of perfusion, prepare 500 ml of 4.0% paraformal...

<ol>
	<li>Hoffman, E. M., Schechter, R., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixative+Composition+Alters+Distributions+of+Immunoreactivity+for+Glutaminase+and+Two+Markers+of+Nociceptive+Neurons,+Nav+1.8+and+TRPV1,+in+the+Rat+Dorsal+Root+Ganglion.">Fixative Composition Alters Distributions of Immunoreactivity for Glutaminase and Two Markers of Nociceptive Neurons, Nav 1.8 and TRPV1, in the Rat Dorsal Root Ganglion.</a> Journal of Histochemistry &#38; Cytochemistry. 58, 329-344 (2010).</li><li>Neves, J. D. S., Omar, N. F., Narvaes, E. A. O., Gomes, J. R., Novaes, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+different+decalcifying+agents+on+EGF+and+EGFR+immunostaining.">Influence of different decalcifying agents on EGF and EGFR immunostaining.</a> Acta Histochemica. 113, 484-488 (2011).</li><li>Watson, R. E., Wiegand, S. J., Clough, R. W., Hoffman, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+cryoprotectant+to+maintain+long-term+peptide+immunoreactivity+and+tissue+morphology.">Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology.</a> Peptides. 7, 155-159 (1986).</li><li>Yen, L. D., Bennett, G. J., Ribeiro-da-Silva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+sprouting+and+changes+in+nociceptive+sensory+innervation+in+the+glabrous+skin+of+the+rat+hind+paw+following+partial+peripheral+nerve+injury.">Sympathetic sprouting and changes in nociceptive sensory innervation in the glabrous skin of the rat hind paw following partial peripheral nerve injury.</a> The Journal of Comparative Neurology. 495, 679-690 (2006).</li><li>Boyette-Davis, J., Xin, W., Zhang, H., Dougherty, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraepidermal+nerve+fiber+loss+corresponds+to+the+development+of+Taxol-induced+hyperalgesia+and+can+be+prevented+by+treatment+with+minocycline.">Intraepidermal nerve fiber loss corresponds to the development of Taxol-induced hyperalgesia and can be prevented by treatment with minocycline.</a> Pain. 152, 308-313 (2011).</li><li>Bloom, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer-Induced+Bone+Remodeling,+Skeletal+Pain,+and+Sprouting+of+Sensory+Nerve+Fibers.">Breast Cancer-Induced Bone Remodeling, Skeletal Pain, and Sprouting of Sensory Nerve Fibers.</a> The Journal of Pain. 12, 698-711 (2011).</li><li>Constantin, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+Tumor+Necrosis+Factor+&#945;+(TNF&#945;)+Requires+TNF+Receptor+Type+2+to+Generate+Heat+Hyperalgesia+in+a+Mouse+Cancer+Model.">Endogenous Tumor Necrosis Factor &#945; (TNF&#945;) Requires TNF Receptor Type 2 to Generate Heat Hyperalgesia in a Mouse Cancer Model.</a> J. Neurosci. 28, 5072-5081 (2008).</li><li>Jankowski, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitization+of+Cutaneous+Nociceptors+after+Nerve+Transection+and+Regeneration:+Possible+Role+of+Target-Derived+Neurotrophic+Factor+Signaling.">Sensitization of Cutaneous Nociceptors after Nerve Transection and Regeneration: Possible Role of Target-Derived Neurotrophic Factor Signaling.</a> The Journal of Neuroscience. 29, 1636-1647 (2009).</li><li>Jimenez-Andrade, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+Sprouting+of+Adult+Nociceptors+in+Chronic+Prostate+Cancer-Induced+Bone+Pain.">Pathological Sprouting of Adult Nociceptors in Chronic Prostate Cancer-Induced Bone Pain.</a> J. Neurosci. 30, 14649-14656 (2010).</li><li>Persson, A. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium-calcium+exchanger+and+multiple+sodium+channel+isoforms+in+intra-epidermal+nerve+terminals.">Sodium-calcium exchanger and multiple sodium channel isoforms in intra-epidermal nerve terminals.</a> Molecular Pain. 6, 84-84 (2010).</li><li>Ohshima, M., Miyake, M., Takeda, M., Kamijima, M., Sakamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+Enterotoxin+B+Causes+Proliferation+of+Sensory+C-Fibers+and+Subsequent+Enhancement+of+Neurogenic+Inflammation+in+Rat+Skin.">Staphylococcal Enterotoxin B Causes Proliferation of Sensory C-Fibers and Subsequent Enhancement of Neurogenic Inflammation in Rat Skin.</a> Journal of Infectious Diseases. 203, 862-869 (2011).</li><li>Johnson, M. S., Ryals, J. M., Wright, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+loss+of+peptidergic+intraepidermal+nerve+fibers+in+an+STZ-induced+mouse+model+of+insensate+diabetic+neuropathy.">Early loss of peptidergic intraepidermal nerve fibers in an STZ-induced mouse model of insensate diabetic neuropathy.</a> Pain. 140, 35-47 (2008).</li></ol>

Here we have detailed the methods for preparation of mouse skin and bone tissue sections for immunostaining and detection of peripheral sensory nerve fibers. The sections produced from plantar punch biopsies contain both glabrous and hairy skin, which means the protocol can be used on any skin type. These techniques can also be employed to stain other cell types in these tissues (e.g. leukocytes, vascular endothelia, smooth muscle among others). These methods provide an excellent compromise between optimal ultrastructura...

<table border="1">
<tbody>
 <tr>
 <td>Material Name</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue No.</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>3mm 			Harris Micro-Punch</td>
 <td>Material</td>
 <td>Ted 			Pella</td>
 <td>15094</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Perfusion 			pump</td>
 <td>Material</td>
 <td>VWR 			International</td>
 <td>23609-170</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Reagent</td>
 <td>Fisher 			Scientific</td>
 <td>T353</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Picric 			acid</td>
 <td>Reagent</td>
 <td>Sigma-Aldrich</td>
 <td>239801</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>OCT 			Embedding compound</td>
 <td>Reagent</td>
 <td>Tissue-Tek</td>
 <td>4583</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cyto-Freeze 			cryogenic aerosol spray</td>
 <td>Material</td>
 <td>Control 			Company</td>
 <td>3118</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Goat 			Serum</td>
 <td>Reagent</td>
 <td>Sigma-Aldrich</td>
 <td>G9023</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Incubation 			tray and lid for Immunostaining (Large)</td>
 <td>Material</td>
 <td>RPI 			Corp.</td>
 <td>248270 			(tray) 
 248270-A 			(lid)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ImmEdge 			hydrophobic barrier pen</td>
 <td>Material</td>
 <td>Vector 			Laboratories</td>
 <td>H-4000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Camel's 			Hair Brushes (#1 thickness)</td>
 <td>Material</td>
 <td>Ted 			Pella</td>
 <td>11859</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pro&#x2010;Long 			Gold Mounting medium</td>
 <td>Reagent</td>
 <td>Invitrogen</td>
 <td>P36930</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

tissue preparation and immunostaining of mouse sensory nerve fibers innervating skin and limb bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Watch this Scientific Journal Video about Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones at JoVE.com

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Research

JoVE Journal

Neuroscience

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...