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% 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 dissolved, cool the solution to room temperature (RT). Slowly add 250 ml of 0.2 M PB while stirring continuously. Using progressively weaker HCl solutions (6 N to 1 N to 0.1 N), adjust the pH to 7.4. Adjust the final volume to 500 ml and chill on ice. Also prepare 400 ml of 0.1 M PB from 0.2 M stock by diluting 1:1 in ddH2O and chill on ice.</li>
	<li>Anesthetize the mouse by intraperitonial injection of an overdose of anesthetic (sodium pentobarbital, 80 mg/kg, administered with a 27G needle).</li>
	<li>During perfusion, 50 ml of 0.1 M PB (dilute 0.2 M PB stock 1:1 with ddH2O) and 150 ml of PFA will be passed sequentially into the mouse circulation. Set up the peristaltic pump by filling the tubing with PB and fixing a 231/2G butterfly needle at one end. Immerse the other end of the tubing into the beaker containing 0.1 M PB. Set the speed of the peristaltic pump to 10 ml/min, and pump through a sufficient amount of the solution to ensure that there are no air bubbles in the tubing.</li>
	<li>Wait until the anesthetized mouse no longer shows any reflex activity, such as toe/tail pinch and blink reflex. Lay the animal on a dissection tray ventral side up inside a fume hood and secure the paws with tape. Spray the fur with 70% ethanol. Make an incision through the skin along the midline to expose the ribcage and the uppermost quarter of the abdominal wall. Then, cut through the abdominal wall at the base of the ribcage. After making this incision, the diaphragm and lower end of the sternum should be visible. Carefully cut from left to right through the margin of the diaphragm, and along the full length of the sternum, taking care not to damage the lungs. Some minor bleeding from the sternum at this stage is normal and will not compromise perfusion. Once the incision along the sternum has been made and the diaphragm cut, it should be possible to lift each half of the ribcage upwards and outwards, such that the heart is exposed. Move aside the lungs if necessary. Both halves of the ribcage can be held in this position with the use of hemostatic clamps. Insert the butterfly needle (231/2G) attached to the peristaltic pump 3-4 mm into the left ventricle, parallel to the long axis of the animal. This placement minimizes the risk of the needle becoming dislodged. Make a small incision in the right atrium to allow the return circulation to flow out of the heart.</li>
	<li>Begin perfusion with 0.1 M PB at 10 ml/min for 4-5 min. If there is still a significant amount of blood emanating from the right atrium at this point, continue until the flow is clear.</li>
	<li>Stop the flow of PB and switch the inlet on the peristaltic pump to the 4% PFA solution. Reduce the flow rate to 5 ml/min and resume pumping PFA solution for 20-25 min. As the PFA enters the circulation, the muscles go into spasm and, after a few minutes, the animal should be literally "fixed" in position. This rigidity can be tested by gently pushing against the hindpaws, taking care not to move the animal and dislodge the cannula. Good perfusion will prevent the limb from flexing in response. Once perfusion with PFA is complete, the cannula and hemostatic clamps can be disconnected and the cadaver prepared for tissue dissection. The dissection approach used depends on the specific tissue.</li>
	<li>Prepare 4% PFA / 5% (v/v) picric acid (PA; for collection and post-fixation of plantar punch only) by adding saturated picric acid solution to 4% PFA. Inclusion of PA significantly improves antigen detectability in peripheral neuronal tissues1</li>
	<li>Prepare bone/cartilage tissue decalcifying/cryoprotectant solution - 10% EDTA in 0.1 M PB with 0.07% glycerol and 15% sucrose. EDTA-based solutions decalcify tissue whilst preserving epitopes2.</li>
	<li>Prepare the cryoprotectant solution - 30% sucrose in 0.1 M PB.</li>
</ol>
2. Tissue Dissection/Removal, Post-fixation and Sectioning
<ol>
	<li>Plantar Punch 
	Place the perfused animal ventral side down on a cutting mat or other sturdy surface. Holding the foot plantar surface-up, press down firmly with the punch biopsy tool (3 mm diameter; Harris micro-punch, Ted Pella Inc.) into the middle of the foot. Turn the biopsy tool back and forth through 180 degrees to confirm the biopsy tool has cut through the entirety of the hind paw. Gently remove the biopsy tool from the foot and eject the tissue into sterile 2 ml tube with cap, containing 1 ml of 4% PFA / 5% PA solution.</li>
	<li>Limb Bone (ex. Femur) 
	Make a lateral incision along the back of the animal at the level of the pelvis, continuing down along both hind limbs. Cut into the pelvis and surrounding muscle to separate the femur from the pelvis whilst leaving the proximal head of the femur intact. Cut into the tibia/fibula to leave the distal head intact, removing the surrounding muscle/periosteum from the bone shaft. Place the femur into a 2 ml tube containing 1 ml of 4% PFA / 5% PA solution.</li>
	<li>Post-fix the tissue samples in 4% PFA / 5% PA solution, with gentle mixing on a rocker for 16-18 h at 4&deg;C</li>
	<li>Decalcify the tissue as follows: For plantar punch (to decalcify the small bones of the foot) place the tissue punch in a sterile 2 ml tube with cap, containing 1.5 ml of 10% EDTA solution in 0.1 M PB with 0.07% glycerol and 15% sucrose. Place the tube on a rocker with mild mixing for 16-18 h at 4&deg;C. For limb bones place the tissue in a sterile 2 ml tube with cap, containing 1.5 ml of 10% EDTA solution in 0.1 M PB with 0.07% glycerol and 15% sucrose. Place the tube on a rocker with mild mixing for 6-7 days at 4&deg;C. The decalcification solution should be changed every 24 hours and the tissue monitored for loss of rigidity with a pair of forceps.</li>
	<li>Transfer the tissue into a sterile 2 ml tube with cap with 1.5 ml of cryoprotectant solution (30% sucrose in 0.1 M PB), and place the tube on a rocker with mild mixing for 16-18 h at 4&deg;C.</li>
	<li>Prepare tissue for sectioning: place a bed volume of optimal cutting temperature (OCT) compound (Sakura Finetek USA Inc.) on a cryostat tissue mounting block and allow to freeze in the cryostat chamber (typically maintained at -20&deg;C). A cryogenic aerosol (Cyto-freeze, Control Co. USA) can also be sprayed on the OCT to accelerate the freezing process. Place tissue specimen on this bed of OCT and cover with an additional thin layer of OCT. Gently spray this OCT with cryogenic aerosol until it has hardened and the tissue within has frozen. Place specimen onto the cutting head in the cryostat chamber and allow the tissue specimen block to equilibrate to the cutting temperature - at least 1 hour. Trying to section when the embedding compound is still too cold results in brittle sections and tissue damage.</li>
	<li>Once the tissue has reached optimal cutting temperature, begin cutting the specimen and generate 40 &mu;m sections.</li>
	<li>For plantar punch collect the cryosections into 12-well tissue culture plates containing 0.1 M PB with 10 mM sodium azide. Make sure that the sections remain submerged in this solution at 4&deg;C. Sections can be stored in this manner for up to 4-6 months for immunostaining purposes. Alternatively, free-floating sections can be stored indefinitely in cryoprotectant solution at -20&deg;C (500 ml 0.1 M PBS, pH 7.2, 30 g sucrose, 10 g PVP40, 300 ml ethylene glycol). Adjust final volume to 1L with distilled water)3. For limb bones collect the cryosections directly onto gelatin pre-coated slides (or Superfrost Plus slides, Fisher Scientific) and allow to air-dry for 1 h, before storing at -20&deg;C. Bone sections on slides stored in this way can be used for immunostaining purposes within 2-3 months.</li>
</ol>
3. Immunostaining of Tissue Sections for Sensory Nerve Fibers
3.1. Plantar punch sections - floating section staining
<ol>
	<li>Using a razor blade, cut 6-7 mm from the end of a 1 ml micropipette tip. Pre-condition the inside of the tip by aspirating 1% fetal bovine serum (FBS) in 0.1 M PB several times (this inhibits sticking of the tissue sections to the walls of the tip). Transfer plantar punch sections to be stained to a 24-well tissue culture plate. Normally 8-10 sections should be stained per well to ensure several high-quality sections are generated per immunostaining.</li>
	<li>Wash plantar punch sections with 500 &mu;l of 0.1 M PB per well for 5 min with vigorous mixing on a rocker at RT. Repeat 2 more times.</li>
	<li>Discard the washing solution and incubate the plantar punch sections in blocking solution, consisting of 10% goat serum and 0.3% Triton X-100 in 0.1 M PB (500 &mu;l of blocking solution per well), with gentle mixing on a rocker for 1 h at 4&deg;C.</li>
	<li>Discard the blocking solution and incubate the tissue sections in primary antibody/antibodies diluted in 250 &mu;l blocking solution for 18-24 h with gentle mixing on a rocker at 4&deg;C. Based on investigator's experimental requirements, single immunolabeling (with one primary antibody against a specific protein or nerve fiber marker), or double immunolabeling (with two primary antibodies against two proteins or nerve fiber markers) can be performed on the same section. While performing double immunolabeling it is important to verify that the two primary antibodies used are raised in different host species (e.g. one raised in mouse and one raised in rabbit), or alternatively, purified monoclonal antibodies of different immunoglobulin G (IgG) isotypes (e.g. one IgG1 and one IgG2a) can be used. It is advisable to seal the plate with Parafilm for overnight incubations to avoid excessive evaporation. In order to stain the peripheral sensory nerve fibers, several specific antibodies can be used. Antibodies against the protein neurofilament 200 (NF200; Sigma) label large-diameter fibers, whereas calcitonin gene-related peptide (CGRP; Sigma) and transient receptor potential vanilloid 1 (TRPV1, Neuromics) label peptidergic, small-diameter peripheral sensory fibers. All peripheral nerve fibers can also be stained with antibodies against &beta;3-tubulin. In skin, collagen IV (Abcam) is used to delineate the basement membrane that separates the epidermis from the dermis. As a general rule, antibodies need to be 5-10 times more concentrated than for cultured cell-based immunofluorescence assays, typically at a working concentration of 5-10 &mu;g/ml.</li>
	<li>Wash the tissue sections with 500 &mu;l of blocking solution (with Triton X-100) per well for 5 min with vigorous mixing on a rocker at RT. Repeat 3 more times.</li>
	<li>Discard the washing solution and incubate tissue sections in appropriate fluorophore-conjugated secondary antibodies (typically diluted 1:1000 in blocking solution, 500 &mu;l) with gentle mixing on a rocker at 4&deg;C. The plate must be wrapped in aluminium foil to avoid photobleaching of fluorophores.</li>
	<li>Wash the tissue sections with 500 &mu;l of blocking solution per well for 10 min with vigorous mixing on a rocker at RT. Then wash with 500 &mu;l of 0.1 M PB with vigorous mixing for 10 min at RT. Finally, wash with 500 &mu;l of 0.05 M PB with vigorous mixing for 10 min at RT.</li>
	<li>Using a 1 ml micropipette with an FBS-treated tip (cut 6-7 mm from the end), aspirate the sections from the tissue culture plate and transfer onto SuperFrost Plus microscope slides. Arrange the sections and absorb excess wash buffer with a fine paintbrush. Avoid prolonged exposure to light. Incubate the slides at RT, in order to allow sections to dry sections on slide (5-30 min).</li>
	<li>Apply several drops of mounting medium (ProLongGold, Vectashield or similar), slowly place a glass coverslip onto sections. Allow the slides to sit in darkness for 5 min, and then seal the coverslip edges with transparent nail polish. Allow air-drying for 15-20 min. The slides can now be visualized or stored at -20&deg;C for several years without any significant loss of fluorescence.</li>
</ol>
3.2. Limb bone sections - on-slide staining
<ol>
	<li>Allow the slides to return to RT from -20&deg;C storage. Using a hydrophobic barrier pen (Super Pap Liquid Blocking Pen, Ted Pella Inc.) circumscribe the region on the slide containing the bone sections to be stained with a generous layer of solution. Allow to air dry for 10-15 min.</li>
	<li>Wash the bone sections with 250 &mu;l of 0.1 M PB per slide for 5 min and repeat for 2 more times.</li>
	<li>Discard the washing solution and incubate the bone sections in blocking solution, consisting of 10% goat serum and 0.3% Triton X-100 in 0.1 M PB (250 &mu;l of blocking solution per slide) for 1 h at 4&deg;C.</li>
	<li>Discard the blocking solution and incubate the bone sections in primary antibody (or combination of primary antibodies, in case of double immunolabeling as mentioned under 3.1.4) diluted in 250 &mu;l blocking solution for 18-24 h at 4&deg;C. It is very important to note that the slides must be placed in a humidified chamber with a sealed lid, in order to avoid drying of primary antibody solution. In order to stain the peripheral sensory nerve fibers, the above-mentioned set of antibodies (see section 3.1.4) can be used with similar working concentrations.</li>
	<li>Wash the tissue sections with 250 &mu;l of blocking solution (with Triton X-100) per slide for 15 min at RT and repeat for 3 more times.</li>
	<li>Discard the washing solution and incubate bone sections in appropriate fluorophore-conjugated secondary antibodies (typically diluted 1:1000 in blocking solution, 250 &mu;l) at 4&deg;C. The staining chamber must be wrapped in aluminium foil to avoid photobleaching of fluorophores. For convenience, it is advisable to use dark-colored staining chambers (e.g. Slide incubation tray/box, RPI Corp.).</li>
	<li>Wash the bone sections with 250 &mu;l of blocking solution per slide for 15 min at RT. Then wash with 500 &mu;l of 0.1 M PB for 15 min at RT. Finally, wash with 500 &mu;l of 0.05 M PB for 15 min at RT. Dip briefly in ddH2O to rinse slides and then incubate at RT in order to allow the bone sections to air dry (5-30 min). Avoid prolonged exposure to light.</li>
	<li>Apply several drops of mounting medium (ProLongGold or similar) and slowly place a glass coverslip onto sections. Let the slides stand in darkness for 5 min, and then seal the coverslip edges with transparent nail polish. Allow drying for 15-20 min. Slides can be stored at -20&deg;C for several years without loss of fluorescence.</li>
</ol>
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.
<img alt="Figure 1" src="/files/ftp_upload/3485/3485fig1.jpg" /> 
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). &beta;3-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 &mu;m); B, D, E and F are confocal image composites generated from an 11-image z-stack taken at 2 &mu;m increments under a 60X objective (scale bar - 50 &mu;m).
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.
<img alt="Figure 2" src="/files/ftp_upload/3485/3485fig2.jpg" /> 
Figure 2. shows immunostaining with anti-CGRP (A-B, red; arrows), anti-NF200 (C-D, red; arrows), anti-&beta;3-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 &mu;m); B, D, E and F are confocal image composites generated from an 11-image z-stack taken at 2 &mu;m increments under a 60X objective (scale bar - 50 &mu;m).

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No conflicts of interests declared.

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 ...

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JoVE Journal

Neuroscience

23.1K Views.  The University of Iowa. تحديد Immunocytochemical الطرفية من الألياف العصبية الحسية فرعية (والكشف عن بروتين تعبير فيها) هي المفتاح لفهم الآليات الجزيئية الكامنة وراء الإحساس الطرفية. نحن هنا تصف طرق تحضير عينات الأنسجة المحيطية / الحشوية ، مثل العظام والجلد أطرافهم ، لimmunostaining محددة من الألياف ا...

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

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron 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. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

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 of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

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 development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

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

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 cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

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 describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

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 isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

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 these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

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

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...