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

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. Video LinkThe 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 for 30 sec or until it approaches boiling. Add 20 g of granular paraformaldehyde (PFA) to the beaker with constant stirring under a fume hood. Add 5 ml of 5 N NaOH drop-wise and stir until the solution clears. When the PFA is completely di...

Induction of thermal and mechanical hypersensitivity by parathyroid hormone–related peptide through upregulation of TRPV1 function and trafficking

“…In addition, the acidic microenvironment of bone-metastasized tumors could provide a favorable condition for constitutive activation of TRPV1 and sustained AP firing, thereby inducing peripheral transmission of painful signals in these pathologies. Our results show PTH1R expression in TRPV1-positive sensory neurons in L4–L6 mouse DRGs that innervate the hind limb bones [36], wherein TRPV1 has also been shown to be expressed on CGRP-positive sensory afferents [77]. PTHrP has been shown to be critical for tumor-induced increase in osteoclast activity [61,86] that constitutes a significant source of the elevated levels of G-CSF/GM-CSF, which also sensitize TRPV1 and nociceptive afferents [76,81].…”

“…Lumbar DRGs (L4–L6) were harvested from adult female C57BL/6 mice perfused with 4% paraformaldehyde (PFA) for 20 min, as described previously [47,77]. The DRGs were stored in 4% PFA with 5% picric acid overnight and were then transferred into 15% sucrose solution in 0.1 M phosphate buffer (PB) for 24 hours.…”

The C-Type Natriuretic Peptide Induces Thermal Hyperalgesia through a Noncanonical Gβγ-dependent Modulation of TRPV1 Channel

“…25 μm-thick sections of fixed DRG tissue were obtained using a cooled cryostat (CM3050 S, Leica Microsystems, Buffalo Groves, IL), as detailed previously (Shepherd and Mohapatra, 2012). Free-floating DRG tissue sections were washed in a 48-well tissue culture plate with 0.1 M PB, before being incubated with blocking/permeabilization solution (10% goat serum + 0.3% Triton X-100 in 0.1 M PB) at 4°C for 60 min.…”

“…The sections were subsequently washed first with 10% goat serum in 0.1 M PB for 5 min, then with 0.1M PB for 5 min, and finally with 0.05M PB for 5 min (all at room temperature with vigorous agitation). Finally, the sections were then carefully transferred onto glass slides and covered with glass coverslips with ProLong Gold antifade reagent (Life Technologies), as described previously (Shepherd and Mohapatra, 2012). Confocal fluorescence images of mounted DRG tissue sections were taken using a BX61WI microscope equipped with the Fluoview 300 laser-scanning confocal imaging system (Olympus) with a 10× objective (NA 0.25; Olympus).…”

Nociceptive TRP Channels: Sensory Detectors and Transducers in Multiple Pain Pathologies

“…Furthermore, TRPV1-3 have also been shown to be expressed in human dental pulp [9,11]. TRPV1 protein expression has been detected in peptidergic sensory afferents innervating bones [77], although expression of other nociceptive TRP channels there remains unexplored. Significant overlap between TRPV1 and TRPA1 expression in small/medium-diameter nociceptive neurons in the DRG and TG has been observed.…”