Name: localization of the locus coeruleus in the mouse brain
Uploaded: 2019-03-07T14:00:00
Description: Watch this Scientific Journal Video about Localization of the Locus Coeruleus in the Mouse Brain at JoVE.com

We thank Abigael Muchenditsi for the maintenance of the mouse colony. Use of the Advanced Photon Source at Argonne National Laboratory was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract number: DE-AC02-06CH11357. We thank Olga Antipova and Dr. Stefan Vogt for user support and assistance at the Advanced Photon Source. This work was funded by the National Institute of Health grant 2R01GM101502 to SL.

Studies of animals was approved by Johns Hopkins University Animal Care and use (ACUC) protocol number M017M385.
1. Brain Slicing
<ol>
	<li>To immobilize, anesthetize mice by the application of 3% isoflurane.
	<ol>
		<li>Soak a cotton ball with drops of isoflurane and place it in a 15 mL microcentrifuge tube. Place the animal&#8217;s nose into the tube and allow it to inhale the isoflurane. Check for the depth of anesthesia by the lack of response to toe-pinch.</li>
	</ol>
	</li>
	<li>Place ...

<ol>
	<li>Robertson, S. D., Plummer, N. W., de Marchena, J., Jensen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+origins+of+central+norepinephrine+neuron+diversity.">Developmental origins of central norepinephrine neuron diversity.</a> Nature neuroscience. 16, 1016-1023 (2013).</li><li>Kobayashi, R. M., Palkovits, M., Jacobowitz, D. M., Kopin, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+mapping+of+the+noradrenergic+projection+from+the+locus+coeruleus.+A+model+for+studies+of+brain+neuronal+pathways.">Biochemical mapping of the noradrenergic projection from the locus coeruleus. A model for studies of brain neuronal pathways.</a> Neurology. 25, 223-233 (1975).</li><li>Olson, L., Fuxe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+projections+from+the+locus+coeruleus+noradrealine+neurons:+the+cerebellar+innervation.">On the projections from the locus coeruleus noradrealine neurons: the cerebellar innervation.</a> Brain research. 28, 165-171 (1971).</li><li>Costa, A., Castro-Zaballa, S., Lagos, P., Chase, M. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+reactions+of+sensomotor+cortex+neurons+to+stimulation+of+emotionally+significant+brain+structures].">Synaptic reactions of sensomotor cortex neurons to stimulation of emotionally significant brain structures].</a> Zhurnal vysshei nervnoi deiatelnosti imeni I P Pavlova. 29, 1248-1257 (1979).</li><li>File, S. E., Deakin, J. F., Longden, A., Crow, T. 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M., Tuppy, M., Moreira, T. S., Takakura, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+locus+coeruleus+catecholaminergic+neurons+in+the+chemosensory+control+of+breathing+in+a+Parkinson's+disease+model.">Role of the locus coeruleus catecholaminergic neurons in the chemosensory control of breathing in a Parkinson's disease model.</a> Experimental neurology. , 172-180 (2017).</li><li>Zarow, C., Lyness, S. A., Mortimer, J. A., Chui, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+loss+is+greater+in+the+locus+coeruleus+than+nucleus+basalis+and+substantia+nigra+in+Alzheimer+and+Parkinson+diseases.">Neuronal loss is greater in the locus coeruleus than nucleus basalis and substantia nigra in Alzheimer and Parkinson diseases.</a> Archives of neurology. 60, 337-341 (2003).</li><li>Chandley, M. 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=Gene+expression+deficits+in+pontine+locus+coeruleus+astrocytes+in+men+with+major+depressive+disorder.">Gene expression deficits in pontine locus coeruleus astrocytes in men with major depressive disorder.</a> Journal of psychiatry &amp; neuroscience : JPN. 38, 276-284 (2013).</li><li>Bernard, R., 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=Altered+expression+of+glutamate+signaling,+growth+factor,+and+glia+genes+in+the+locus+coeruleus+of+patients+with+major+depression.">Altered expression of glutamate signaling, growth factor, and glia genes in the locus coeruleus of patients with major depression.</a> Molecular psychiatry. 16, 634-646 (2011).</li><li>Gos, 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=Tyrosine+hydroxylase+immunoreactivity+in+the+locus+coeruleus+is+elevated+in+violent+suicidal+depressive+patients.">Tyrosine hydroxylase immunoreactivity in the locus coeruleus is elevated in violent suicidal depressive patients.</a> European archives of psychiatry and clinical neuroscience. 258, 513-520 (2008).</li><li>Bielau, H., 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=Immunohistochemical+evidence+for+impaired+nitric+oxide+signaling+of+the+locus+coeruleus+in+bipolar+disorder.">Immunohistochemical evidence for impaired nitric oxide signaling of the locus coeruleus in bipolar disorder.</a> Brain research. 1459, 91-99 (2012).</li><li>Wiste, A. K., Arango, V., Ellis, S. P., Mann, J. J., Underwood, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norepinephrine+and+serotonin+imbalance+in+the+locus+coeruleus+in+bipolar+disorder.">Norepinephrine and serotonin imbalance in the locus coeruleus in bipolar disorder.</a> Bipolar disorders. 10, 349-359 (2008).</li><li>Borodovitsyna, O., Flamini, M. D., Chandler, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Stress+Persistently+Alters+Locus+Coeruleus+Function+and+Anxiety-like+Behavior+in+Adolescent+Rats.">Acute Stress Persistently Alters Locus Coeruleus Function and Anxiety-like Behavior in Adolescent Rats.</a> Neuroscience. 373, 7-19 (2018).</li><li>Hirschberg, S., Li, Y., Randall, A., Kremer, E. J., Pickering, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+dichotomy+in+spinal-+vs+prefrontal-projecting+locus+coeruleus+modules+splits+descending+noradrenergic+analgesia+from+ascending+aversion+and+anxiety+in+rats.">Functional dichotomy in spinal- vs prefrontal-projecting locus coeruleus modules splits descending noradrenergic analgesia from ascending aversion and anxiety in rats.</a> eLife. 6, (2017).</li><li>McCall, J. G., 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=CRH+Engagement+of+the+Locus+Coeruleus+Noradrenergic+System+Mediates+Stress-Induced+Anxiety.">CRH Engagement of the Locus Coeruleus Noradrenergic System Mediates Stress-Induced Anxiety.</a> Neuron. 87, 605-620 (2015).</li><li>Borges, G. P., Mico, J. A., Neto, F. L., Berrocoso, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticotropin-Releasing+Factor+Mediates+Pain-Induced+Anxiety+through+the+ERK1/2+Signaling+Cascade+in+Locus+Coeruleus+Neurons.">Corticotropin-Releasing Factor Mediates Pain-Induced Anxiety through the ERK1/2 Signaling Cascade in Locus Coeruleus Neurons.</a> The international journal of neuropsychopharmacology. 18, (2015).</li><li>Simone, 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=Ethinyl+estradiol+and+levonorgestrel+alter+cognition+and+anxiety+in+rats+concurrent+with+a+decrease+in+tyrosine+hydroxylase+expression+in+the+locus+coeruleus+and+brain-derived+neurotrophic+factor+expression+in+the+hippocampus.">Ethinyl estradiol and levonorgestrel alter cognition and anxiety in rats concurrent with a decrease in tyrosine hydroxylase expression in the locus coeruleus and brain-derived neurotrophic factor expression in the hippocampus.</a> Psychoneuroendocrinology. 62, 265-278 (2015).</li><li>Carter, M. 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=Tuning+arousal+with+optogenetic+modulation+of+locus+coeruleus+neurons.">Tuning arousal with optogenetic modulation of locus coeruleus neurons.</a> Nature. 13, 1526-1533 (2010).</li><li>Fan, Y., 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=Corticosterone+administration+up-regulated+expression+of+norepinephrine+transporter+and+dopamine+beta-hydroxylase+in+rat+locus+coeruleus+and+its+terminal+regions.">Corticosterone administration up-regulated expression of norepinephrine transporter and dopamine beta-hydroxylase in rat locus coeruleus and its terminal regions.</a> Journal of neurochemistry. 128, 445-458 (2014).</li><li>Xiao, 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=Copper+regulates+rest-activity+cycles+through+the+locus+coeruleus-norepinephrine+system.">Copper regulates rest-activity cycles through the locus coeruleus-norepinephrine system.</a> Nature chemical biology. 14, 655-663 (2018).</li><li>Amaral, D. G., Sinnamon, H. 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+locus+coeruleus:+neurobiology+of+a+central+noradrenergic+nucleus.">The locus coeruleus: neurobiology of a central noradrenergic nucleus.</a> Progress in neurobiology. 9, 147-196 (1977).</li><li>Ralle, 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=Disease+at+a+Single+Cell+Level:+intracellular+copper+trafficking+activates+compartment-specific+responses+in+hepatocytes.">Disease at a Single Cell Level: intracellular copper trafficking activates compartment-specific responses in hepatocytes.</a> The Journal of Biological Chemistry. 285, 30875-30883 (2010).</li><li>Paxinos, G., Franklin, K. B. 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Phys. IV France. 104, 635-638 (2003).</li><li>Davies, K. 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=Copper+pathology+in+vulnerable+brain+regions+in+Parkinson's+disease.">Copper pathology in vulnerable brain regions in Parkinson's disease.</a> Neurobiology of aging. 35, 858-866 (2014).</li><li>Davies, K. M., Mercer, J. F., Chen, N., Double, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper+dyshomoeostasis+in+Parkinson's+disease:+implications+for+pathogenesis+and+indications+for+novel+therapeutics.">Copper dyshomoeostasis in Parkinson's disease: implications for pathogenesis and indications for novel therapeutics.</a> Clinical science. 130, 565-574 (2016).</li><li>James, S. 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Properly orienting the specimen is a crucial step in this protocol. Because we are using anatomical features of the dorsal surface of the brain to locate LC (boundary between cerebellum and inferior colliculus), it is important that the sections be aligned properly. This requires care in properly setting the brain into the mouse brain slicer matrix. We recommend cutting ~500 &#956;m more tissue anterior and posterior to LC to avoid missing the nucleus. The most common mistake is to cut too few sections that results in mi...

The locus coeruleus (LC) is an important region in the brainstem and a major site of norepinephrine (NE) production1. The LC sends projections throughout the brain2 to the cortex, the hippocampus and the cerebellum3 and regulates major physiological processes, including circadian rhythm4,5, attention and memory6, stress7, cognitive processes8, and emotion9,10. Dysfunction of LC has been implicated in neurological and neuropsychiatric disorders11, including Parkinson disease12,13,14, Alzheimer disease14, depression15,16,17, bipolar disorder18,19, and anxiety20,21,22,23,24. Given these roles, analysis of LC is crucial to studying its function and dysfunction.
Mice are widely used for studies of physiologic and pathophysiologic processes. Due to their small size, the mouse LC has an average diameter of ~300 &#956;m, leading to difficulty locating the structure. During brain sectioning, the LC can easily be missed in either coronal or sagittal sections. Available studies describing identification of LC in animals do not offer a step-by-step protocol that a non-expert can follow1,25. Thus, to offer guidance for the localization of LC, we describe a protocol that we developed to locate this region in the mouse brain for several applications (Figure 1, Figure 2, Figure 3). The protocol applies carefully controlled brain sectioning and immunohistochemical detection of DBH26,27, or alternatively TH24, both enzymes highly enriched in the LC28. Once LC is located by immunohistochemistry, adjacent brain slices can be used for further studies, including morphological and metabolic analyses, as well as metal imaging studies via X-ray fluorescence microscopy (XFM)29. We describe XFM as an example in this protocol (Figure 3).

<table>
	<tbody>
		<tr>
			<td>Adult mouse brain slicer matrix</td>
			<td>Zivic Instruments</td>
			<td>BSMAS001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit secondary antibody, Alexa Fluor 488 (source - donkey)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Charged glass slides</td>
			<td>Genesee</td>
			<td>29-107</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Microm GmbH</td>
			<td>HM 505E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat cutting blades</td>
			<td>Thermo Fisher Scientific</td>
			<td>MX35</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors Mini, 9.5cm</td>
			<td>Antech Diagnostcs</td>
			<td>503241</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopamine &#946;-hydroxylase (DBH) antibody - inhouse production (source - rabbit)</td>
			<td>B. Eipper</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopamine &#946;-hydroxylase (DBH) antibody - commercially availabe (source - rabbit)</td>
			<td>Cell Signaling</td>
			<td>8586</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes, 50ml</td>
			<td>USA Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>Forane (isofluorane)</td>
			<td>Baxter</td>
			<td>NDC 1019-360-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Micro Adson</td>
			<td>Antech Diagnostcs</td>
			<td>501245</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardset mounting media</td>
			<td>EM sciences</td>
			<td>17984-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Pascal</td>
			<td>LSM 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-well plates, 24 wells</td>
			<td>Thermo Fisher Scientific</td>
			<td>930186</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal cutting temperature compound (OCT)</td>
			<td>VWR/ tissue tech</td>
			<td>102094-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)/ formalin 10%</td>
			<td>Fisher Scientific</td>
			<td>SF98-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Peel-A-Way disposable embedding molds</td>
			<td>Polysciences Inc.</td>
			<td>18646A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencil brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Tech</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Amazon</td>
			<td colspan="2">ASIN: B000CMFJZ2</td>
		</tr>
		<tr>
			<td>Spatulas</td>
			<td>Antech Diagnostcs</td>
			<td>14374</td>
			<td></td>
		</tr>
		<tr>
			<td>T pins</td>
			<td>Office Depot</td>
			<td>344615</td>
			<td></td>
		</tr>
		<tr>
			<td>The Mouse Brain in Stereotaxic Coordinates, Paxinos and Franklin, 3rd Edition</td>
			<td>Amazon</td>
			<td>ISBN: 978-0123694607</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100 (to prepare PBSD)</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosine hydroxylase (TH) antibody (source - rabbit)</td>
			<td>EMD Millipore</td>
			<td>AB152</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultralene thin film for XRF</td>
			<td>SPEX Sample Prep</td>
			<td>3525</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-field fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Axio Zoom.V16</td>
			<td></td>
		</tr>
	</tbody>
</table>

localization of the locus coeruleus in the mouse brain

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional abnormalities of LC impact several brain regions including cortex, hippocampus, and cerebellum and may contribute to depression, bipolar disorder, anxiety, as well as Parkinson disease and Alzheimer disease. These disorders are often associated with metal misbalance, but the role of metals in LC is only partially understood. Morphologic and functional studies of LC are needed to better understand the human pathologies and contribution of metals. Mice are a widely used experimental model, but the mouse LC is small (~0.3 mm diameter) and hard to identify for a non-expert. Here, we describe a step-by-step immunohistochemistry-based protocol to localize the LC in the mouse brain. Dopamine-&#946;-hydroxylase (DBH), and alternatively, tyrosine hydroxylase (TH), both enzymes highly expressed in the LC, are used as immunohistochemical markers in brain slices. Sections adjacent to LC-containing sections can be used for further analysis, including histology for morphological studies, metabolic testing, as well as metal imaging by X-ray fluorescence microscopy (XFM).

Changes in metal homeostasis (such as Cu, Fe, Zn, and Mn) are often observed in neurologic disorders, including changes in the LC34,35. Thus, determining metal levels in the brain is necessary for understanding of disease mechanisms. The brain sections generated using the described protocol can be used to quantify the levels of Cu and other metals in the LC and compare them to the levels in regions outside of the LC. (<strong clas...

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Localization of the Locus Coeruleus in the Mouse Brain

The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus.

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional ...

Properly orienting the specimen is a crucial step in this protocol. Because we are using anatomical features of the dorsal surface of the brain to locate LC (boundary between cerebellum and inferior colliculus), it is important that the sections be aligned properly. This requires care in properly setting the brain into the mouse brain slicer matrix. We recommend cutting ~500 &#956;m more tissue anterior and posterior to LC to avoid missing the nucleus. The most common mistake is to cut too few sections that results in missing the LC entirely. Thus, for one&#8217;s first time following this protocol, we recommend cutting more sections than necessary. Careful study of the brain atlas images prior to staining is very helpful. The appearance of the brainstem changes appreciably every few hundred microns and, with some experience, it is possible to know what sections are worth staining simply by the macroscopic appearance.

During the process of localizing the LC, there might be variations in the signal depending on how well the brain was oriented during sectioning. When cutting through the center of the LC, the signal is bright and covers a larger area as compared to the edge of the LC, which will show up as a signal over a much smaller area. In the case that coronal slices are slightly tilted, the LC of one side of the 4th ventricle might be apparent and the one on the other side might only be visible in an adjacent slide. Thus, one cannot always expect the appearance of both LC regions at maximum intensity within one brain slice. This artifact can be avoided by cutting the brain exactly coronal in the mouse brain slicer matrix and carefully embedding the brain into the cubical embedding mold with OCT.

Immunostaining, at least with the anti-tyrosine hydroxylase antibody, is extremely forgiving and, in our experience, works on sections up to 100 &#956;m in thickness. We have found that blocking solution is not necessary for high signal-to-noise staining of LC, reducing cost and reducing the amount of time needed to locate LC. In our experience, the staining protocol can be sped up &#8211; albeit with reduced quality and penetrance of staining &#8211; by reducing permeabilization to 2 h, primary antibody for 8 h, and secondary antibody for 2 h. Additionally, if one is simply interested in locating LC (e.g., for validating injection of a virus/tracer), sections from a fixed brain can be cut on a vibratome at 100 &#956;m thickness.

One limitation of this protocol is it, by design, requires euthanizing the animal and removing the brain. Therefore, it is not useful for in vivo localization (e.g., electrophysiologic recordings). Another limitation is that this protocol requires PFA fixation which might alter the native state of the tissue. These alterations include the elemental content such as copper, calcium, iron and zinc36. The actual alteration of metal distribution caused by PFA fixation may be tested in one sample which can be run in parallel to a non-fixed sample. A comparison of the metal distribution between these two samples will provide evidence on the effect of PFA fixation on the distribution of the metal which is of interest in a certain study. If PFA fixation must be avoided, the general principle of this protocol (locating LC by immunostaining and using adjacent sections for follow-up experiments) can be extended to frozen sections without fixation.

We note that this protocol is largely a refinement of existing methods to solve this problem. The novelty exists in tailoring previous approaches to locate a very small, easily missed nucleus. We expect that this protocol can be easily modified and extended based on need (e.g., using transgenic animals expressing fluorophores in LC to avoid immunostaining).

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