The authors would like to thank Dr. Louis Reichardt for the TrkAtaulacZ mice and Dr. Annette Markus for insightful discussion and suggestions. This work was supported by startup funds from the Burke Foundation as well as Whitehall Foundation research grant 2010-08-61, a research grant from Wings for Life Foundation (WFL-US-028/14), grant ZB1-1102-1 from the Christopher &#38; Dana Reeve Foundation, and grants 1R01EY022409 and 3R01EY022409-01S1 from the National Eye Institute, to JZ. KJO is a Goldsmith fellow.

NOTE: All procedures comply with the NIH Guide for the Use and Care of Laboratory Animals. The animal protocol was approved by the IACUC at Weill Cornell Medical College.
1. Tissue Preparation
<ol>
	<li>Euthanize timed-pregnancy females by cervical dislocation15. Dissect embryonic E16 - E18 embryos from timed-pregnancy females and place embryos individually in the wells of a 6-well dish, filled with cold phosphate buffered saline (PBS).</li>
	<li>Rinse embryos in cold PBS.</li>
	<li>Remove a small portion of the amniotic membrane of the embryo and place in a pre-prepared proteinase K solution for subsequent genotyping. Alternatively, if the amniotic membrane is lost, remove a small portion of the tip of the embryo tail and place it in proteinase K solution</li>
	<li>Poke holes into the skin all around the embryo using insect pins (at least 100 per side).</li>
	<li>Remove PBS and slowly add fresh 2% paraformaldehyde (PFA), pH 7.4, to the well.</li>
	<li>Gently shake for 2 hr at 4 &#176;C.</li>
	<li>Rinse embryos twice in PBS and store in PBS at 4 &#176;C until staining.</li>
</ol>
2. Genotyping
<ol>
	<li>Immerse amniotic membranes or embryo tails in proteinase K mixture according to the manufacturer&#8217;s instructions.</li>
	<li>Incubate at 56 &#176;C for 10 min.</li>
	<li>Extract DNA using a commercially available DNA purification kit.</li>
	<li>Take 0.5 &#181;l of the total 200 &#181;l purified genomic DNA solution, combine it with 0.5 &#181;l of each of the gene-specific sense and antisense primers (10 &#181;M), 2 &#181;l dNTP Mix (2.5 mM of dATP, dCTP, dGTP and dTTP), 0.2 &#181;l Taq polymerase (5 U/&#181;l) and 2 &#181;l PCR buffer (10x), add H2O to a total volume of 20 &#181;l. 
	NOTE: Primer sequences are as follows (Table 1): 
	TrkA: TrkA_F: GCG GGC GCC GCC GCG ATG, TrkA_R: GAA GCC GCC TGC GCG GCT CTG CCA GGG TG; PCR fragment length: 400 basepairs (bp). 
	Bax: In5R: GTT GAC CAG AGT GGC GTA GG, Ex5F: TGA TCA GAA CCA TCA TG, NeoR: CCG CTT CCA TTG CTC AGC GG; PCR fragment lengths: wild type, 304 bp; Bax null, 507 bp. 
	TauLacZ: lacZup: ACA ACG TCG TGA GTG GGA AAA, lacZdown: ATC AAC ATT AAA TGT GAG CGA G; PCR fragment length: 360 bp.</li>
	<li>Run a PCR with the following program for TrkA: step 1: 1 min at 95 &#176;C, step 2: 10 sec at 95 &#176;C, step 3: 20 sec at 68 &#176;C, step 4: 30 sec at 72 &#176;C. Repeat steps 2 - 4 for 40 cycles.
	<ol>
		<li>For LacZ and Bax, run a PCR with the following program: step 1: 1 min at 95 &#176;C, step 2: 10 sec at 95 &#176;C, step 3: 1 min at 54 &#176;C, step 4: 1 min at 72 &#176;C, repeat steps 2 - 4 for 35 cycles.</li>
	</ol>
	</li>
	<li>Run PCR samples on a 2% agarose gel at 100 V in 1x Tris-Acetate-EDTA buffer for 20 min with a lane of DNA ladder to assess fragment lengths.</li>
	<li>Analyze embryos for the presence of wild type TrkA, TrkA-tauLacZ and Bax null alleles. The genotypes of experimental embryos are TrkAtaulacZ/WT : Bax -/- and TrkAtaulacZ/taulacZ : Bax-/-, depending on the experimental questions. Embryos lacking the tauLacZ reporter will be negative for axonal staining and can therefore serve as negative controls to calibrate the staining process.</li>
</ol>
3. Embryo Staining
<ol>
	<li>Use a commercial kit for lacZ staining with some modifications as described below in sections 3.2 - 3.8. Here, use the Millipore kit.</li>
	<li>Immerse embryos in Buffer A for at least 1 hr, shaking gently at room temperature.</li>
	<li>Directly immerse embryos in Buffer B for at least 15 min, shaking gently at room temperature.</li>
	<li>While embryos are in Buffer A/B, pre-warm Tissue Base solution at 37 &#176;C. Use 5 ml of Tissue Base solution per embryo.</li>
	<li>While embryos are in Buffer A/B, prepare fresh 5-bromo-&#8203;4-&#8203;chloro-&#8203;3-&#8203;indolyl &#945;-D-galactopyranoside (X-gal) at a concentration of 40 mg/ml in 100% dimethyl sulfoxide (DMSO).</li>
	<li>Dilute X-gal into the pre-warmed Tissue Base solution at a concentration of 1 mg/ml and add 5 ml of X-gal/Tissue Base mixture to a glass scintillation vial.</li>
	<li>Transfer embryos to a glass scintillation vial containing X-gal/Tissue Base mixture. Seal lid with Parafilm and incubate at 37 &#176;C overnight, checking for presence of blue stain.</li>
	<li>Postfix embryos with fresh 4% PFA, pH 7.4. Gently shake for 4 hr at 4 &#176;C.</li>
	<li>Rinse embryos twice in cold PBS.</li>
</ol>
4. Tissue Dehydration and Clearing
<ol>
	<li>Place embryos in 50% methanol/ 50% PBS mixture for 30 min at room temperature, shaking in the glass vials.</li>
	<li>Continue to dehydrate in 75% methanol/ 25% PBS for 30 min at room temperature, shaking in glass vials.</li>
	<li>Continue to dehydrate in 90% methanol/ 10% PBS for 30 min at room temperature, shaking in glass vials.</li>
	<li>Continue to dehydrate in 100% methanol for 30 min (2x) at room temperature, shaking in glass vials.</li>
	<li>While embryos are dehydrating, prepare a 1:1 (v:v) mixture of benzoyl alcohol and benzoyl benzoate. 
	NOTE: Benzoyl alcohol and benzoyl benzoate is toxic.</li>
	<li>Immerse embryos in the 1:1 (v:v) mixture of 100% methanol: 100% BABB for 15 min. Only use glass because the BABB will dissolve plastic.</li>
	<li>Immerse embryos in 100% BABB to completely clear the tissue and to visualize X-gal staining in the cleared embryo. 
	NOTE: Image as soon as possible because the X-gal stain will fade over time.</li>
</ol>
5. Whole Mount Imaging
<ol>
	<li>Stabilize the embryo, immersed in 100% BABB, at the appropriate angles for photography using metal forceps. Illuminate using a combination of up-light and under-light light sources.</li>
	<li>Acquire images with a dissecting microscope outfitted with a digital camera. Take multiple bright field exposures at five successive focal planes 0.5 mm apart along the Z axis. Exposure times and f-stops may vary according to illumination and camera specs and need to be optimized for each setup.</li>
	<li>Use standard image processing software to process overlay images and generate photomontages.</li>
</ol>

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The authors declare that there are no competing interests.

The above-described X-gal staining procedure of embryonic TrkAtaulacZ mice allows for the rapid and detailed visualization of long distance axon projections in the intact fixed embryo. Because of the Bax null background these mice allow for the probing of signaling mechanisms that may contribute to both axon growth and neuronal survival. Mating with transgenic or knockout mice of interest allows for comprehensive assessment of axonal phenotypes and can serve as useful guide for futur...

Establishment of precise neuronal networks is a complex developmental process essential for the functionality of the nervous system. Disturbance in this process leads to neuronal dysfunction which has been implicated in human neurological diseases1-3. To study the underlying molecular mechanisms of axon growth and target innervation in mammals, we have developed a protocol to visualize the axonal trajectories of TrkA-expressing sensory neurons using a combination of two genetically modified mouse lines.
TrkA is a receptor for nerve growth factor NGF and is a functional marker of nociceptive sensory neurons4. TrkA is highly expressed in nociceptive neurons during early development and mediates NGF-dependent neuron survival, axon growth, arborization and target innervation5-9. In TrkAtaulacZ mice, the wild type TrkA gene is replaced by a taulacZ expression cassette10, such that the axonal morphology of putative TrkA-positive neurons can be visualized by &#946;-gal (X-gal) staining11. Using a heterozygous TrkAtaulacZ/WT line, we can examine factors that may regulate or interfere with the development of sensory afferent projections in vivo.
Moreover, TrkA expression is absent in homozygous TrkAtaulacZ/taulacZ mice, which can therefore be used to assess the axon growth promoting mechanisms in the absence of NGF/TrkA signaling. Since nociceptive neurons depend on NGF/TrkA signaling not only for axon growth, but also for survival, we employ another mouse line, lacking the pro-apoptotic Bax gene, to inhibit apoptosis in embryonic DRG neurons, rescuing them from cell death that is otherwise observed in the absence of TrkA signaling. The Bax-/- background12 thus allows for the molecular dissection of signaling pathways that specifically affect axon growth7-9,13-15. In TrkA-/- : Bax-/- mice, DRG neurons survive, but sensory afferent innervation in the skin is completely abolished14,15. We can selectively activate signaling pathways to determine their respective contributions to the development of axon projections. The utility of this method is that it allows the assessment of changes in axonal growth phenotypes when different genetic modifications are bred onto the TrkAtaulacZ/taulacZ : Bax-/- or TrkAtaulacZ/WT : Bax-/- backgrounds.

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;"></td>
			<td style="width:127px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Tech</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution A</td>
			<td>Millipore</td>
			<td>BG-6-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution B</td>
			<td>Millipore</td>
			<td>BG-7-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Stain Base Solution</td>
			<td>Millipore</td>
			<td>BG-8-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintiallation vial</td>
			<td>Kimble Chase</td>
			<td>74500-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Labline</td>
			<td>Model 120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>FST</td>
			<td>26000-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well dish</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td>IDT</td>
			<td>custom DNA primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara dNTP mixture</td>
			<td>Takara</td>
			<td>4030</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Takara LA buffer</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara LA Taq</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR machine</td>
			<td>Bio-Rad&#160;</td>
			<td>DNA Engine Dyad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>B-1042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B-6630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>M205A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Leica</td>
			<td>DFC310FX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring light&#160;</td>
			<td>Leica&#160;</td>
			<td>MEB110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photoshop</td>
			<td>Adobe</td>
			<td>Photoshop 4.0</td>
		</tr>
	</tbody>
</table>

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.

The genotypes of TrkAWT/taulacZ : Bax-/- and TrkAtaulacZ/taulacZ : Bax-/- embryos can be unambiguously determined by standard PCR genotyping (Figure 1). X-gal staining displays detailed peripheral axonal arbors subcutaneously in conventionally stained embryos (Figures 2, 3a), and throughout the embryo after tissue clearing (Figures 3b, 4).
We have bred the TrkAWT/taulacZ : Bax-/-</sup...

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

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 ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

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Neuroscience

10.0K Views.  Weill Medical College of Cornell University. 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.

Video: Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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 Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...