This work was supported by research grants (NIH/NIDCD 009269, 012831 and ARRA administrative supplement NIH grants) to Weihong Lin. We especially thank Mr. Tim Ford at UMBC for his technical assistance in videotaping and processing. We also wish to thank Dr. Daphne Blumberg, Ms. Chere Petty at UMBC and Mr. Nicholas McCollum from Olympus America Inc. for their equipment assistance in videotaping.

1. Mouse Nose Preparation
We used adult C57BL/6 background mice in this study. All animal care and procedures are approved by the Animal Care and Use Committees (IACUC) of University of Maryland, Baltimore County.

 1.1 Acquiring the nose from paraformaldahyde-fixed mice
<img src="/files/ftp_upload/50538/50538fig1.jpg" alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50538/50538fig1highres.jpg"/> 
Figure 1. Bones of a mouse skull. A: Dorsal view of the skull. B: Ventral view of the skull with the mandible removed. The skull was prepared from a 40-day old mouse. Individual bones are colored for better visualization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50538/50538fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<ol>
 <li>Perfuse transcardially to fix individual mice following the protocol of Lin, et al., (2008)16. Briefly, mice were deeply anesthetized with tribromoethanol (Avertin 250 &mu;g/g body weight), perfused transcardially with 0.1M phosphate buffer (PB, 30-50 ml), followed by a phosphate buffered fixative containing 3% paraformaldehyde, 19 mM L-lysine monohydrochloride, and 0.23% sodium m-periodate (approximately 35-50 ml). One can also follow the steps in the JoVE article for animal perfusion 35. </li>
 <li>Use a pair of scissors to cut off mandible (or the lower jaw) and remove the skin on the head. </li>
 <li>Separate the entire head from the rest of the body. </li>
 <li>Remove the palate. Also, clean and remove the remaining connective tissue and muscle on the surface of the skull to obtain the specimen shown in Figures 1A and 1B.</li>
 <li>Under a dissection microscope, remove the skull bone covering the brain and olfactory bulbs. Trim off excess tissue and bones. Note, for the extended tissue preparation in which the brain and nose remain connected, only the skull bones are removed. For immunohistochemical experiments, the tissue was post-fixed for 1.5 hr and transferred to 0.1M phosphate buffered saline (PBS) with 25% sucrose overnight. Nasal tissue should be kept humidified throughout the dissection by dipping it in the buffered sucrose solution several times. </li>
</ol>
1.2 Acquiring the nose from freshly euthanized mice
<ol>
 <li>Individual mice were transferred to a clean cage and exposed to CO2 gas, which was followed by cervical dislocation 5 min after the final breath. To reduce the blood in the nose tissue, use a pair of scissors to open the chest and cut the heart to allow blood to drain. </li>
 <li>Repeat steps 1.1.2 to 1.1.5, except the post-fixation and cryoprotection with 25% sucrose. The specimen should be kept humidified and maintained with Tyrode&#x2019;s saline containing (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 Na pyruvate, 10 D-glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (HEPES, adjusted at pH 7.4). Alternatively, fold a piece of Kimwipes, soak it with Tyrode&#x2019;s solution, and place beneath the specimen while dissecting and occasionally dip the specimen in the Tyrode's solution or drop some solution onto the tissue to keep it humidified to maintain the viability of the cells and tissues. </li>
</ol>
2. Incisor, Anterior Vomer and Maxilla Bone Removal
<ol>
 <li>Start from a ventral viewpoint. Locate the vomer bone and break the vomer bone along its length using a rongeur or forceps with teeth to break the ventral most part of the bone. </li>
 <li>Using serrated forceps, remove the broken segments of the vomer bone by gently plying the bone fragments away from the vomeronasal organ (VNO). Note, for mice less than a month old, or if only interested in the MOE, these two steps can be skipped.</li>
 <li>Hold the head firmly with forceps. Use the rongeur to break the front portion of both incisors and the jointed region of anterior maxilla between the two incisors and the vomer bone. </li>
 <li>Break the ventral portion of the right maxilla at the region just anterior to the zygomatic arch up to the level of the dorsal zygomatic plate. </li>
 <li>Flip over the nose for a dorsal viewpoint. Use the rongeur to break the right maxilla anterior of the dorsal zygomatic plate. The entire right maxilla anterior of the zygomatic plate should be loose. Use the fine forceps to gently separate any nasal tissue underlying the maxilla, and then gently lift away the bone fragment. </li>
 <li>Repeat steps 2.4 and 2.5 to loosen the left incisor and left anterior maxilla and remove them after the nasal bone has been removed. </li>
</ol>
3. Nasal Bone and Dorsal Zygomatic Plate Removal 
<ol>
 <li>Use the serrated forceps or the rongeur to remove the remainder of the frontal bones just caudal to the nasal bones. After removing this piece of bone, the caudal part of the nasal bone can be gripped using forceps.</li>
 <li>Use the rongeur to break the anterior portion of the zygomatic arch, which is connected to the zygomatic plate. </li>
 <li>Take the fine forceps at the lateral edge of the dorsal end of the zygomatic plate and gently flip the bone and remove it. If the bone is not loose, use the rongeur to clamp it gently to loosen it for removal. </li>
 <li>Use the fine forceps or a razor blade to loosen the medial suture between the right and left nasal bones. </li>
 <li>Use the serrated forceps to grip the caudal end of the right nasal bone. Gently move the bone from side to side to separate it from underlying tissue. It is useful to move the forceps along the caudal third of the nasal bone for moving the bone side to side. As the nasal bone separates, slowly lift the bone from the caudal end. </li>
 <li>While the nasal bone is slightly lifted, tilt the bone laterally to reveal a lateral outcropping of the bone that is lined with thin respiratory epithelial tissue. Use fine forceps to gently release this tissue from the nasal bone. Continue to lift the nasal bone. When the bone is completely separated from underlying tissue, use scissors to cut off the nasal bone at the rostral end. </li>
 <li>Repeat steps 3.1, 3.5 and 3.6 for the removal of left nasal bone.</li>
 <li> Repeat steps 3.2 and 3.3 for the left side of the nose. </li>
</ol>
4. Lateral Zygomatic Plate Removal 
<ol>
 <li>Remove the zygomatic plate one side at a time. Either zygomatic plate can be removed first.</li>
 <li>From a ventral viewpoint, break the maxilla inferior to the zygomatic arch until break reaches the zygomatic arch. </li>
 <li>From a dorsal viewpoint, gently grab the zygomatic arch and lift forward and lateral. If the zygomatic plate is still attached to any tissue, take the fine forceps and gently sever the connections between the tissue and the bone. </li>
 <li>Repeat for the zygomatic plate on the other side of the nose. </li>
</ol>
5. Orbit Bone Removal
<ol>
 <li>From a ventral viewpoint, break the palantine bone between the molars of the nose. </li>
 <li>Use the rongeur to break the 3 molars and the maxilla on each side of the nose. </li>
 <li>Break and remove any remaining thick pieces of bone ventral and posterior to the turbinates on each side of the nose. </li>
</ol>
6. Ethmoid Bone Removal
<ol>
 <li>Break any portion of the ethmoid bone protruding caudal to the turbinates. This is necessary to avoid the loss of turbinate tissue when removing thin pieces of the ethmoid bone covering the turbinates.</li>
 <li>For the right side of the nose, place the fine forceps at the anterior edge of the ethmoid bone and gently remove it. If a portion of the bone remains, repeat this procedure until all the thin bone covering the turbinates has been removed. The turbinate bones do not need to be removed in most of the preparations. In aged mice, the cribriform plate becomes brittle. If cryosectioning of the nasal tissue is needed, remove small pieces of the plate with fine forceps to reduce the potential damage caused by the bone. </li>
 <li>Repeat step 6.2) for the left side of the nose. </li>
 <li>Remove any remaining bone fragments prior to sectioning. Note: in animals more than a year-old, the posterodorsal region of the septum bone is somewhat thick and hard. One can remove this portion using fine forceps. Insert the tip of the forceps into both side of the bone to separate the dorsal portion of the septum bone and the lining epithelial tissue. Use one pair of forceps to grab the bone and hold the specimen. Use another pair of forceps to break the upper bony portion from the lower cartilaginous portion of the septum and gently remove it. </li>
</ol>
7. Nose Preparation for Cryosectioning
<ol>
 <li>Set up the aspirator vacuum pump. </li>
 <li>Place the nose in an embedding mold. Submerge the nose in OCT media.</li>
 <li>Use a vacuum to remove air bubbles trapped within the nose tissue. This process takes up to 5 min.</li>
 <li>After removing air bubbles, set the tissue in the desired orientation.</li>
 <li>Freeze the OCT and tissue in the mold using dry ice. The embedded tissue can then be cryosectioned immediately or stored at -80 &deg;C for future use. </li>
</ol>

<ol>
	<li>Naclerio, R. M., Pinto, J., Assanasen, P., Baroody, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+ability+of+the+nose+to+warm+and+humidify+inspired+air.">Observations on the ability of the nose to warm and humidify inspired air.</a> Rhinology. 45, 102-111 (2007).</li><li>Bjermer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nose+as+an+air+conditioner+for+the+lower+airways.">The nose as an air conditioner for the lower airways.</a> Allergy. 54, 26-30 (1999).</li><li>Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+olfactory+system+makes+sense+of+scents.">How the olfactory system makes sense of scents.</a> Nature. 413, 211-218 (2001).</li><li>Bryant, B., Silver, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemisthesis: The common chemical sense. , (2000).</li><li>Gross, E. A., Swenberg, J. A., Fields, S., Popp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+morphometry+of+the+nasal+cavity+in+rats+and+mice.">Comparative morphometry of the nasal cavity in rats and mice.</a> J. Anat. 135, 83-88 (1982).</li><li>Halpern, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+and+function+of+the+vomeronasal+system.">The organization and function of the vomeronasal system.</a> Annu. Rev. Neurosci. 10, 325-362 (1987).</li><li>Rodolfo-Masera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Su+l'esquoestizenza+di+un+particulare+organo+olfacttivo+nel+setto+nasale+della+cavia+e+di+altri+roditori.">Su l'esquoestizenza di un particulare organo olfacttivo nel setto nasale della cavia e di altri roditori.</a> Arch. Ital. Anat. Embryol. 48, 157-212 (1943).</li><li>Levai, O., Strotmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+pattern+of+nerve+fibers+from+the+septal+organ:+DiI-tracing+studies+with+transgenic+OMP+mice.">Projection pattern of nerve fibers from the septal organ: DiI-tracing studies with transgenic OMP mice.</a> Histochemistry and Cell biology. 120, 483-492 (2003).</li><li>Storan, M. J., Key, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Septal+organ+of+Gruneberg+is+part+of+the+olfactory+system.">Septal organ of Gruneberg is part of the olfactory system.</a> J. Comp. Neurol. 494, 834-844 (2006).</li><li>Restrepo, D., Arellano, J., Oliva, A. M., Schaefer, M. L., Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+views+on+the+distinct+but+related+roles+of+the+main+and+accessory+olfactory+systems+in+responsiveness+to+chemosensory+signals+in+mice.">Emerging views on the distinct but related roles of the main and accessory olfactory systems in responsiveness to chemosensory signals in mice.</a> Horm. Behav. 46, 247-256 (2004).</li><li>Breer, H., Fleischer, J., Strotmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sense+of+smell:+multiple+olfactory+subsystems.">The sense of smell: multiple olfactory subsystems.</a> Cell Mol. Life Sci. 63, 1465-1475 (2006).</li><li>Munger, S. D., Leinders-Zufall, T., Zufall, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsystem+organization+of+the+mammalian+sense+of+smell.">Subsystem organization of the mammalian sense of smell.</a> Annu. Rev. Physiol. 71, 115-140 (2009).</li><li>Finger, T. E., St Jeor, V. L., Kinnamon, J. C., Silver, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+substance+P-+and+CGRP-immunoreactive+nerve+fibers+in+the+nasal+epithelium+of+rodents.">Ultrastructure of substance P- and CGRP-immunoreactive nerve fibers in the nasal epithelium of rodents.</a> J. Comp. Neurol. 294, 293-305 (1990).</li><li>Papka, R. E., Matulionis, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+substance-P-immunoreactive+nerves+with+the+murine+olfactory+mucosa.">Association of substance-P-immunoreactive nerves with the murine olfactory mucosa.</a> Cell Tissue Res. 230, 517-525 (1983).</li><li>Baraniuk, J. N., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasonasal+reflexes,+the+nasal+cycle,+and+sneeze.">Nasonasal reflexes, the nasal cycle, and sneeze.</a> Curr. Allergy Asthma Rep. 7, 105-111 (2007).</li><li>Lin, W., Ogura, T., Margolskee, R. F., Finger, T. E., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM5-expressing+solitary+chemosensory+cells+respond+to+odorous+irritants.">TRPM5-expressing solitary chemosensory cells respond to odorous irritants.</a> J. Neurophysiol. 99, 1451-1460 (2008).</li><li>Ogura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+microvillous+cells+in+the+mouse+main+olfactory+epithelium+and+effect+of+acetylcholine+on+olfactory+sensory+neurons+and+supporting+cells.">Cholinergic microvillous cells in the mouse main olfactory epithelium and effect of acetylcholine on olfactory sensory neurons and supporting cells.</a> J. Neurophysiol. 106, 1274-1287 (2011).</li><li>Finger, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solitary+chemoreceptor+cells+in+the+nasal+cavity+serve+as+sentinels+of+respiration.">Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 8981-8986 (2003).</li><li>Gulbransen, B. D., Clapp, T. R., Finger, T. E., Kinnamon, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+solitary+chemoreceptor+cell+responses+to+bitter+and+trigeminal+stimulants+in+vitro.">Nasal solitary chemoreceptor cell responses to bitter and trigeminal stimulants in vitro.</a> J. Neurophysiol. 99, 2929-2937 (2008).</li><li>Zhao, K., Dalton, P., Yang, G. C., Scherer, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+modeling+of+turbulent+and+laminar+airflow+and+odorant+transport+during+sniffing+in+the+human+and+rat+nose.">Numerical modeling of turbulent and laminar airflow and odorant transport during sniffing in the human and rat nose.</a> Chemical Senses. 31, 107-118 (2006).</li><li>Ressler, K. J., Sullivan, S. L., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+zonal+organization+of+odorant+receptor+gene+expression+in+the+olfactory+epithelium.">A zonal organization of odorant receptor gene expression in the olfactory epithelium.</a> Cell. 73, 597-609 (1993).</li><li>Vassar, R., Ngai, J., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+segregation+of+odorant+receptor+expression+in+the+mammalian+olfactory+epithelium.">Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium.</a> Cell. 74, 309-318 (1993).</li><li>Fulle, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+receptor+guanylyl+cyclase+expressed+specifically+in+olfactory+sensory+neurons.">A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 92, 3571-3575 (1995).</li><li>Juilfs, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subset+of+olfactory+neurons+that+selectively+express+cGMP-stimulated+phosphodiesterase+(PDE2)+and+guanylyl+cyclase-D+define+a+unique+olfactory+signal+transduction+pathway.">A subset of olfactory neurons that selectively express cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D define a unique olfactory signal transduction pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. 94, 3388-3395 (1997).</li><li>Lin, W., Arellano, J., Slotnick, B., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odors+detected+by+mice+deficient+in+cyclic+nucleotide-gated+channel+subunit+A2+stimulate+the+main+olfactory+system.">Odors detected by mice deficient in cyclic nucleotide-gated channel subunit A2 stimulate the main olfactory system.</a> The Journal of Neuroscience: The Official journal of the Society for Neuroscience. 24, 3703-3710 (2004).</li><li>Ishii, T., Omura, M., Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+two-+and+three-color+fluorescent+RNA+in+situ+hybridization+of+the+main+and+accessory+olfactory+epithelia+in+mouse.">Protocols for two- and three-color fluorescent RNA in situ hybridization of the main and accessory olfactory epithelia in mouse.</a> J. Neurocyt. 33, 657-669 (2004).</li><li>Lee, A. C., Tian, H., Grosmaitre, X., Ma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+odorant+receptors+and+response+properties+of+olfactory+sensory+neurons+in+aged+mice.">Expression patterns of odorant receptors and response properties of olfactory sensory neurons in aged mice.</a> Chemical Senses. 34, 695-703 (2009).</li><li>Packard, A., Schnittke, N., Romano, R. A., Sinha, S., Schwob, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeltaNp63+regulates+stem+cell+dynamics+in+the+mammalian+olfactory+epithelium.">DeltaNp63 regulates stem cell dynamics in the mammalian olfactory epithelium.</a> The Journal of Neuroscience: the official journal of the Society for Neuroscience. 31, 8748-8759 (2011).</li><li>Matthews, J. B., Mason, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+decalcifying+agents+on+immunoreactivity+of+formalin-fixed,+paraffin-embedded+tissue.">Influence of decalcifying agents on immunoreactivity of formalin-fixed, paraffin-embedded tissue.</a> Histochem J. 16, 771-787 (1984).</li><li>Athanasou, N. A., Quinn, J., Heryet, A., Woods, C. G., McGee, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+decalcification+agents+on+immunoreactivity+of+cellular+antigens.">Effect of decalcification agents on immunoreactivity of cellular antigens.</a> J. Clin. Pathol. 40, 874-878 (1987).</li><li>Hegg, C. C., Irwin, M., Lucero, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+store-mediated+signaling+in+sustentacular+cells+of+the+mouse+olfactory+epithelium.">Calcium store-mediated signaling in sustentacular cells of the mouse olfactory epithelium.</a> Glia. 57, 634-644 (2009).</li><li>Spehr, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+the+main+olfactory+system+in+social+recognition+of+major+histocompatibility+complex+peptide+ligands.">Essential role of the main olfactory system in social recognition of major histocompatibility complex peptide ligands.</a> The Journal of Neuroscience: the official journal of the Society for Neuroscience. 26, 1961-1970 (2006).</li><li>Ma, M., Chen, W. R., Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+characterization+of+rat+and+mouse+olfactory+receptor+neurons+from+an+intact+epithelial+preparation.">Electrophysiological characterization of rat and mouse olfactory receptor neurons from an intact epithelial preparation.</a> J. Neurosci. Methods. 92, 31-40 (1999).</li><li>Cygnar, K. D., Stephan, A. B., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+responses+of+mouse+olfactory+sensory+neurons+using+the+air-phase+electroolfactogram+recording.">Analyzing responses of mouse olfactory sensory neurons using the air-phase electroolfactogram recording.</a> J. Vis. Exp. (37), e1850 (2010).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J. Vis. Exp. (65), e3564 (2012).</li><li>Lin, W., Margolskee, R., Donnert, G., Hell, S. W., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+neurons+expressing+transient+receptor+potential+channel+M5+(TRPM5)+are+involved+in+sensing+semiochemicals.">Olfactory neurons expressing transient receptor potential channel M5 (TRPM5) are involved in sensing semiochemicals.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 2471-2476 (2007).</li><li>Lin, W., Ezekwe, E. A., Zhao, Z., Liman, E. R., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM5-expressing+microvillous+cells+in+the+main+olfactory+epithelium.">TRPM5-expressing microvillous cells in the main olfactory epithelium.</a> BMC Neurosci. 9, 114 (2008).</li><li>Ogura, T., Krosnowski, K., Zhang, L., Bekkerman, M., Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoreception+regulates+chemical+access+to+mouse+vomeronasal+organ:+role+of+solitary+chemosensory+cells.">Chemoreception regulates chemical access to mouse vomeronasal organ: role of solitary chemosensory cells.</a> PLoS One. 5, e11924 (2010).</li><li>Sathyanesan, A., Feijoo, A. A., Mehta, S. T., Nimarko, A. F., Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profile+of+G-protein+&#946;&#947;+subunit+gene+transcripts+in+the+mouse+olfactory+sensory+epithelia.">Expression profile of G-protein &#946;&#947; subunit gene transcripts in the mouse olfactory sensory epithelia.</a> Frontiers in Cellular Neuroscience. 7, 84 (2013).</li></ol>

The authors declare that they have no competing financial interests.

Here, we demonstrated a step-by-step procedure for isolating intact olfactory and respiratory tissue from the mouse nose by sequentially removing the surrounding bones while sparing the tissue below. We show that careful bone removal can preserve even the most delicate tissues in their entirety. We also share insight into possible modifications of this technique, in which we isolate both the brain and nose tissue together to preserve the nerve connection. This new method provides a means for isolating whole olfactory and respiratory tissues in a single specimen for further processing in immunohistochemical, RNA in situ hybridization, and physiological experiments.
Advantages of Bone Removal
Before our method, decalcifying agents were typically used to soften bones for tissue sectioning and immunohistochemistry. However, depending on the size of the bone and the age of the animals, decalcification can be a lengthy process requiring tissue incubation in bone decalcifiers for hours or even several days29,30. The agents commonly used also contain acids, which may have effects on the tissue that can alter or impede immunolabeling 29,30. To combat this, some researchers use ethylenediaminetetraacetic acid (EDTA) solution to sequester calcium ions from the bone 24,26-28. This method of decalcification also requires days. The dissection shown here can be performed in about 20-30 min and does not require the application of any chemical solutions that may alter the stainability of tissue. 
Our method can also be used to obtain intact nasal tissue from freshly dissected noses, providing an advantage for obtaining live tissue for physiological recordings. For example, nose slices are commonly used for Ca2+ imaging of OSNs and supporting cells in the olfactory epithelium 31,32. These slices have to be prepared from neonatal mice before the bones surrounding the tissues hardened. However, neonatal specimens are not identical to adults because the olfactory epithelium continues development and maturation postnatally. Using our method, researchers can obtain intact olfactory tissue from mice older than neonatal ages. This presents a significant advantage, especially in studies of age-dependent changes. 
Modifications and Troubleshooting
We showed one effective sequence to remove surrounding bones. However, there are other sequences of bone removal to obtain intact nasal tissues, and the step by step procedure may be modified depending on the individual needs of the researcher and the tissues of most interest. For example, if the vomeronasal organ is needed, the vomer bone should be removed early in the procedure because there are more bone-covered areas to grasp to hold the tissue in the desired place during removal. By adapting the procedure to each application, tissues of interest are more likely to remain intact for further processing. The nasal bones also tend to be difficult to remove, as the tissue below clings to the bones and will often tear as the bones are lifted away. In addition to wiggling the nasal bones as shown in the video, small forceps may be gently inserted between the nasal bone and the underlying tissue to help move the tissue down and away from the bones during removal. 
The dissection also varies slightly depending on the age of the mice. In young mice, the bones tend to be softer, while the bones of older mice are more brittle and strongly fused to neighboring bones in some regions. Soft bone is easier to separate and to remove in small pieces than harder bone. This difference can be easily overcome by using forceps to score or scrape at connections between bones to aid in their separation and clean removal. In older animals, the posterodorsal region of septum is somewhat thick and hard and should be removed prior to cryosectioning. The bony portion of the septum can be removed as described in procedure 6.4. Although there are small differences in the dissection for older and younger mice, age is not a limiting factor. In our studies, we have successfully prepared intact nasal tissues from mice ranging from 14 days to two years old. 
There is no significant difference between the dissecting steps used for fixative-fixed nose and for fresh nose although the fresh tissue is softer and requires more careful manipulation. In both preparations, keeping the tissue humidified during the dissection is important. It is not necessary to perform the fresh tissue dissection in buffered saline, however, periodically dipping the tissue in Tyrode&#x2019;s solution or dripping the solution onto the tissue while dissecting is critical as it will keep the tissue humidified and nutrient supplied to maintain its viability.
The procedure presented here can be further modified to include bone removal that leaves both the brain and nose intact in a single specimen (Figure 2B). This modification spares the nerve connections between the brain and nose that pass through the cribriform plate. More technical skill and time are required for this dissection than the nose alone. However, preserving the connections from the periphery to the brain allows for more diverse applications of this method. 
Limitations
We have used this method to prepare nasal tissues for a number of studies, including immunohistochemical labeling and mRNA in situ hybridization analysis, and have successfully labeled many proteins in signaling pathways, stained cell markers in various cell populations, and examined morphological features and cell distribution through the nasal cavity16,17,36-39. We did not experience limitations in cryosectioning and immunolabeling. For studies that require cryosectioning of the extended preparation that keeps the brain and nasal tissue connected in a single specimen, we recommend to use mice younger than 4 months since in older mice the brittle cribriform plate may create tissue damage during sectioning. However, for fresh tissue intended for electrophysiological recordings, the medial tissues between turbinates are not readily accessible in the whole mount preparation. Removal of small pieces of tissue or splitting in discrete regions may overcome the problem. Because conventional electro-olfactogram is done on the medial surface of endo-turbinates 25,33,34, our tissue preparation may allow recordings from other regions with little manipulation. 
Applications beyond Immunohistochemistry
Many applications of the procedure exist for those who have mastered the technique. Possible applications include zonal studies on olfactory and respiratory tissues which, using other methods, have been difficult to obtain intact and in their original anatomical configuration. Applications also exist for electrophysiology by allowing simultaneous multiple-recordings on whole, fresh tissues. Some of these applications, which were difficult to perform using existing techniques, are made possible by our deboning method. Further, this protocol can also be adapted to biochemical, genomic, and proteomic studies that require consistent sample collection at various regions for critical comparison.
In summary, we have developed a procedure to rapidly and reliably access olfactory and respiratory tissues in the mouse nose by removing the surrounding bones. This method has significant advantages over commonly used techniques such as decalcification. Further, our method requires no chemical treatment; therefore, tissues are not limited for use in immunohistochemistry experiments, but also can be applicable for in situ hybridization and physiological studies. Thus, our method is a faster, more direct way to prepare the mouse nose for further processing. We expect our method will facilitate olfactory and respiratory studies in nasal regions.

The mammalian nasal cavity contains various types of tissues and organs that serve distinct functions. The nasal cavity makes up the entry portion of the upper respiratory tract, which allows air travel into and out of the lungs. Inhaled air passes through the nasal cavity where it undergoes temperature and humidity conditioning 1 as well as cleaning or filtering to remove irritating and toxic substances and infectious microorganisms 2. Both treatments are carried out by nasal epithelia and subepithelial tissues, including glands and vessels and are critical for protecting the lower airways and the lungs. In addition to its role in respiration and epithelial defense, the nasal tissue also contains peripheral sensory apparatuses of the olfactory and trigeminal systems, which detect a wide range of chemical substances in the passing air. Depending on which system is activated, sensory detection of chemicals in the nose can elicit either a sense of smell, irritation, or pain 3,4. 
The peripheral olfactory system is complex and made up of several anatomically separated olfactory sensory organs within the nasal cavity. Among them, the main olfactory epithelium (MOE) is the largest, which makes up approximately 45-52% of the nasal epithelia in rodents 5 and is located in the posterior region. In the anteroventral region, there is a pair of tubular structures known as the vomeronasal organ 6, which sit along each side of the nasal septum. Two additional small groupings of olfactory sensory neurons, known as the septal organ of Masera 7,8 and the Gruneberg ganglion 9, reside along the ventral septum and the dorsal entry region of the nasal cavity, respectively. These peripheral organs contain neuro-epithelia with distinctive features in morphology, cell marker expression, and physiological function. Together they detect thousands of odor molecules with exquisite sensitivity 10-12. 
In addition to the olfactory sensory organs, the nasal cavity also houses other sensory systems. It is known that peptidergic trigeminal nerve fibers are present in the nasal epithelium, especially the respiratory epithelium 13,14. Some of these fibers detect irritating and toxic chemicals and are responsible for initiating protective reflexes such as coughing and sneezing 4,15. Irritating odorous and bitter compounds can also be detected by a recently discovered population of solitary chemosensory cells (SCCs), many of which are innervated by trigeminal nerve fibers 16-19. These SCCs are located in higher density in the entry region of the nasal cavity and vomeronasal entry ducts, hinting that they may also serve a protective function 16-18. Thus, nasal epithelia can differ substantially in function, morphology, and cell composition depending on their anatomical locations.
Even within a single and specialized epithelium, there are regional differences. The MOE is one such example. The MOE lines various turbinates, which are complicated and curled structures. Because of them, different regions of the MOE experience different air flow rates, and thus, different diffusion and clearance rates of airborne odor molecules 20. Also, it is known that olfactory sensory neurons (OSNs) expressing a given odor receptor are located in one of four circumvented zones of the MOE 21,22. How this location difference affects an OSN&#x2019;s response to odorants is largely not known. In addition, some OSN populations exhibit regional preference. Guanylyl cyclase-D (GC-D)-expressing OSNs have zonal distributions favoring the cul-de-sac regions of the ectoturbinates 23,24. More recently, we found a subpopulation of canonical OSNs that expresses transient receptor potential channel M5 (trpM5) and is preferentially located in the lateral and ventral regions 25. These results indicate that MOE is not uniform. However, how these regional differences affect olfactory coding is not understood. This is in part because thorough physiological investigation of the MOE and the nose has been limited by the difficulty of obtaining intact nasal epithelia with preserved anatomical organization using current methods.
The nasal epithelia are predominantly surrounded by the anterior bones of the skull, including the nasal, maxilla, palatine, zygomatic, and ethmoid bones. In adult mice and other rodent models, these bones are hard and difficult to remove without damaging the closely associated nasal tissue, particularly the delicate turbinates. Often, chemical-based decalcification is used to soften bones to allow cryosectioning of nasal tissues for morphological, immunohistochemical, and in situ hybridization studies; however, depending on the age of the animal, the decalcification process can last overnight up to 7 days 24,26-28. This treatment is also limited because it requires tissue be fixative-preserved. Additionally, chemical decalcification can be harsh and affect the immunolabeling of some sensitive antibodies 29,30. For physiological studies, live tissue is required, and thus, these experiments are often conducted on isolated OSNs or MOE slices obtained from neonates whose skull bones are thin and soft 17,31,32. Physiological studies can also utilize whole mount preparations by splitting the head 25,33,34, but usually only the medial surface of the nose is easily accessible, limiting physiological recordings on other areas. 
Here, we describe an effective, manual deboning method to prepare intact nasal tissues with preserved original anatomical organization and morphology. We sequentially remove the major bones of the anterior skull under a dissection microscope to expose an almost entirely intact nasal epithelium while keeping the thin turbinate bones intact unless the mice are very old and cryosectioning is needed. We also extend the method to preserve the connection between the nasal tissues and olfactory bulbs, as well as the rest of the brain, thus facilitating simultaneous examination of both peripheral and central circuits. Our method can be used to prepare paraformaldehyde-fixed, as well as fresh, live nasal tissue. Thus, our method is expected to facilitate morphological, immunohistochemical and physiological studies of respiration, olfaction, and nasal damage and illness.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Dissection&lt;/strong&gt;</td></tr><tr><td>Rongeur, 1.0 mm Jaw width</td><td>World Precision Instruments (WPI)</td><td>501333</td><td></td></tr><tr><td>Fine forceps, Dumont 3 </td><td>WPI</td><td>503235</td><td></td></tr><tr><td>Fine forceps, Dumont 55</td><td>WPI</td><td>14099</td><td></td></tr><tr><td>Fine forceps, Dumont AA</td><td>Fine Science Tools (FST)</td><td>11210-20</td><td></td></tr><tr><td>Specimen forceps, Serrated</td><td>VWR</td><td>82027-440</td><td></td></tr><tr><td>Operating scissors</td><td>WPI</td><td>501753</td><td></td></tr><tr><td>Iris scissors, Straight</td><td>Miltex</td><td>V95-304</td><td></td></tr><tr><td>Dissection microscope</td><td>Olympus</td><td>SZ40</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Tissue embedding&lt;/strong&gt;</td></tr><tr><td>Optimum cutting temperature (OCT) compound</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>Plastic embedding mold</td><td>VWR</td><td>15160-215</td><td></td></tr><tr><td>Aspirator vacuum pump</td><td>Fisher Scientific</td><td>09-960-2</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Section staining &lt;/strong&gt;</td></tr><tr><td>Neutral red</td><td>ACROS Organic</td><td>CAS 553-24-2</td><td>Nuclei staining</td></tr></tbody></table>

an effective manual deboning method to prepare intact mouse nasal tissue with preserved anatomical organization

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

Using this method, we can reliably obtain almost entirely intact nasal tissue. Figure 2A shows an image of adult nasal specimen from a paraformaldehyde-fixed head. In this specimen, all four sub-olfactory sensory organs, including the MOE, septal organ, the Gruneberg ganglion, and VNO, are intact. Also, the respiratory epithelia and subepithelial tissues, such as glands and vessels, are preserved. We have successfully used this method in a number of studies in which we investigated morphology, distribution, nerve innervation, and cell marker expression in various specialized sensory cell populations 16,17,36-38. 
Figure 2B shows a specimen with the brain, olfactory bulbs, and nose together. This extended preparation is especially useful in studies that require nerve connection between the peripheral and central olfactory systems. We prepared this specimen by removing the skull bones surrounding the brain prior to the removal of facial bones. Our extended method allows investigators to conduct experiments on both peripheral and central olfactory system in a single preparation. 
The nasal tissue shown in Figure 2 can be used for whole-mount observation, as well as for preparation of tissue sections. Figure 3A show a horizontal section cut through both the MOE and respiratory regions. Figure 3B shows a coronal section cut through the middle of the MOE. We stained the sections with neutral red for a better view of various types of epithelial and submucosal structures at different anatomical locations. These results demonstrate that our technique preserves even the delicate MOE turbinate structure and anatomical organization of the nose, which enable comprehensive and comparative investigation of nasal morphological and functional changes under normal and pathological conditions. 
<img src="/files/ftp_upload/50538/50538fig2.jpg" alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50538/50538fig2highres.jpg"/> 
Figure 2. Isolated nasal and brain tissue. A: Dorsal view of intact nose tissue dissected using our deboning method. B: Dorsal view of a nose and brain with neural connections between the central and peripheral olfactory systems intact. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50538/50538fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img src="/files/ftp_upload/50538/50538fig3.jpg" alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50538/50538fig3highres.jpg"/> 
Figure 3. Sections of nasal tissues. A: A horizontal section showing olfactory and respiratory epithelia as well as other nasal structures. B: Coronal section through the turbinates of the MOE in the posterior of the nose. Both sections were 14 &mu;m-thick and stained with neutral red. MOE: Main olfactory epithelium. RE: Respiratory epithelium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50538/50538fig3large.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization at JoVE.com

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

an-effective-manual-deboning-method-to-prepare-intact-mouse-nasal

Nanyang Technological University

Research

JoVE Journal

Biology

35.1K Views.  University of Maryland Baltimore County. An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

Video: An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Neuroscience

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.

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 ...

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Developmental Biology

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

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 ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Immunology and Infection

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

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

Whole Animal Perfusion Fixation for Rodents

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain