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 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.
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.
2. Tissue Dissection/Removal, Post-fixation and Sectioning
3. Immunostaining of Tissue Sections for Sensory Nerve Fibers
3.1. Plantar punch sections – floating section staining
3.2. Limb bone sections – on-slide staining
4. Representative Results
4.1. Plantar punch sections
Plantar punch tissue sections can be visualized under epifluorescence microscope, or under a confocal microscope with a 10X, 40X or 63X objective.
Figure 1. A-D show staining with Collagen IV antibody (green) in basement membranes, at the epidermal-dermal junction and in cartilage and muscle. Numerous CGRP- (A-B, red) and NF200-positive fibers (C-D, red) are distributed throughout the mouse skin in the plantar region (arrows). β3-tubulin is a pan-neuronal marker (E, red), whereas TRPV1 staining (F; red) is mainly confined to small-diameter fibers that are also CGRP-positive (green; arrowheads). A and C are epifluorescence images taken with a 10X objective (scale bar -500 μm); B, D, E and F are confocal image composites generated from an 11-image z-stack taken at 2 μm increments under a 60X objective (scale bar – 50 μm).
4.2. Limb bone sections
Limb bone tissue sections can also be visualized under epifluorescence microscope, or under a confocal microscope with a 10X, 40X or 63X objective.
Figure 2. shows immunostaining with anti-CGRP (A-B, red; arrows), anti-NF200 (C-D, red; arrows), anti-β3-tubulin (E; red) and anti-TRPV1 (F; red) co-stained with anti-CGRP antibody (green; arrows). These images show subtypes of sensory nerve fibers distributed throughout the bone matrix in the spongy head region of mouse femur. A and C are epifluorescence images taken with a 10X objective (scale bar -500 μm); B, D, E and F are confocal image composites generated from an 11-image z-stack taken at 2 μm increments under a 60X objective (scale bar – 50 μm).
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 ultrastructural preservation (which is achieved by glutaraldehyde fixation, but frequently results in disruption of epitopes and diminished immunostaining staining quality) and immunocytochemical detectability, if the procedures are followed step-by-step in a rigorous manner.
Detection of sensory nerve fibers in these tissues can aid in our understanding of the regulation of peripheral neurite outgrowth and sprouting4, as well as anatomical changes in peripheral sensory afferents under different pathological conditions. Furthermore, changes in the expression of neurotransmitters, receptors, ion channels or other phenotypic markers in normal developmental or pathological conditions can also be studied3,5-10. Along with appropriate electrophysiological, biochemical and behavioral testing, such changes in peripheral sensory neuron staining patterns can be used to test hypotheses related to various pain states6,9, inflammation11 and neuropathies5,12. In conclusion, these techniques provide an invaluable source of in vivo data that complements and reinforces other anatomical, structural and functional data acquired through additional approaches, furthering our understanding of the regulation and acquisition of plasticity in peripheral sensory nerve fibers in health and disease.
The authors have nothing to disclose.
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.
Material Name | Tipo | Company | Catalogue No. | Comment |
3mm Harris Micro-Punch | Material | Ted Pella | 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 | RPI Corp. | 248270 (tray) 248270-A (lid) |
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ImmEdge hydrophobic barrier pen | Material | Vector Laboratories | H-4000 | |
Camel’s Hair Brushes (#1 thickness) | Material | Ted Pella | 11859 | |
Pro‐Long Gold Mounting medium | Reagent | Invitrogen | P36930 |