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

Summary

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.

Abstract

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.

Protocol

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.

1. 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°C. This will be used for perfusion and post-fixation processes.
2. On the day of perfusion, prepare 500 ml of 4.0% paraformaldehyde in 0.1M PB (PFA, pH 7.4) fixative solution, a volume sufficient for the perfusion of 2 mice: microwave 200 ml of ddH₂O in a glass beaker....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3mm Harris Micro-Punch	Material	Ted Pella, Inc.	15094
Perfusion pump	Material	VWR international	23609-170
Paraformaldehyde	Reagent	Fisher Scientific	T353
Picric acid	Reagent	Sigma-Aldrich	239801
OCT Embedding compound	Reagent	Tissue-Tek	4583
Cyto-Freeze cryogenic aerosol spray	Material	Control Company	3118
Goat Serum	Reagent	Sigma-Aldrich	G9023
Incubation tray and lid for Immunostaining (Large)	Material	Research Products International Corp.	248270 (tray) 248270-A (lid)
ImmEdge hydrophobic barrier pen	Material	Vector Laboratories	H-4000
Camel’s Hair Brushes (#1 thickness)	Material	Ted Pella, Inc.	11859
Pro‐Long Gold Mounting medium	Reagent	Invitrogen	P36930