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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Brain Slicing
<ol>
	<li>To immobilize, and 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&#39;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 the animal on its back and immobilize it by pinning its extremities down with a T pin while having access to its abdomen.</li>
	<li>Cut the animal with surgical scissors, snipping the abdominal skin and cutting through the skin in the thorax region. Pin the skin down using T-pins. Then, break the peritoneal membrane up to the thorax. Expose the heart by cracking the thoracic cavity and cutting the diaphragm.</li>
	<li>Cut the right atrium to allow the blood to flow out of the animal. Insert a 10 mL syringe with a 25-gauge needle into the left ventricle and perfuse with 10 mL of phosphate-buffered saline (PBS). 
	NOTE: This allows the solution to flow through systemic circulation and exit through the right atrium.</li>
	<li>Remove the 10 mL syringe and insert a 25-gauge needle attached to the 60 mL syringe. Perfuse through the left ventricle with 50 mL of ice-cold 4% paraformaldehyde (PFA).
	<ol>
		<li>Prepare ice cold 4% PFA solution by diluting 10% PFA solution in H2O and chilling the final 4% PFA solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Isolate the head of the mouse and remove the brain from the skull.
	<ol>
		<li>Cut the skin from the neck, and then cut towards the eyes to expose the skull. Crack the skull from the neck to the nose, and then from one eyeball to the other. Peel the skull out and excise the whole brain.</li>
	</ol>
	</li>
	<li>Incubate the brain in 4% PFA for 24 h at 4 &#176;C.</li>
	<li>Transfer the brain with forceps into a 50 mL conical tube filled with 25 mL of 30% sucrose solution. Keep it at 4 &#176;C for 48-72 hours until the brain sinks to the bottom of the tube.</li>
	<li>Cut the brain with an adult mouse brain slicer matrix coronal through the midbrain (~3 mm posterior of bregma). Keep the brain section containing the brainstem. 
	NOTE: This will result in two brain sections &#8211; one containing most of cortex (anterior of the cut) and one containing the brainstem/cerebellum (posterior of the cut). Use the brainstem section for the following steps.</li>
	<li>Embed the brainstem section with the cut surface placed on the bottom of an embedding mold, surrounded by optimal cutting temperature compound (OCT); move the embedded brain to a -80 &#176;C freezer and freeze for at least 12 h &#8211; until further use.</li>
	<li>Cutting at the cryostat: place the embedding mold containing the brain in OCT into the cryostat; incubate it in the cryostat for several hours to adjust the temperature of the brain block to that of the cryostat.</li>
	<li>Peel away the embedding mold to expose the OCT block containing the brain.</li>
	<li>Use razor blades to remove excess OCT from the surface of the block without touching the brain.</li>
	<li>Mount the OCT block on the chuck of the cryostat, exposing the cut surface of the brain toward the front.</li>
	<li>Adjust the cut surface of the brain so that it is oriented in parallel to the razorblades of the cryostat.</li>
	<li>Trim the brain beginning at the medulla, cutting 100 &#181;m sections rostrally.</li>
	<li>Trim rostrally until the cerebellum and brainstem are cut as one continuous slice. Begin collecting slices at 50 &#181;m thickness. 
	NOTE: As one trims rostrally from the medulla, the brainstem and cerebellum will be cut as two separate sections. In the rostral sections, the brainstem and cerebellum will eventually merge at the level of the 4th ventricle. Once the lateral edges of the 4th ventricle are well-formed, then the cerebellum and brainstem will come out as one continuous slice.
	<ol>
		<li>Collect an OCT-surrounded brain slice with forceps and place it in a well of a 24-well plate filled with PBS (Figure 1a). The locus coeruleus (LC) will be most prominent when the cerebellum and inferior colliculus meet one another at ~-5.52 mm posterior of bregma (Figure 2b). 
		NOTE: The most anterior part of LC will disappear once the cerebellum has been fully sectioned and no longer surrounds the inferior colliculus at ~-5.34 mm posterior of bregma (Figure 2c).</li>
	</ol>
	</li>
</ol>
2. Immunohistochemistry for Dopamine &#946;-Hydroxylase (DBH) or Tyrosine Hydroxylase (TH) (Figure 1)
<ol>
	<li>Day 1
	<ol>
		<li>Wash the selected slices three times for 5 min in PBS.</li>
		<li>Permeabilize for 24 h in 0.5% phosphate-buffered saline with detergent (PBSD) at 4 &#176;C.
		<ol>
			<li>Dilute 125 &#181;L of detergent in 25 mL of PBS.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Wash slices three times for 5 minutes in 0.5% PBSD.</li>
		<li>Add the primary antibody, anti-DBH or anti-TH, for 18 h at a dilution of 1:500 in 0.5% PBSD at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Wash slices three times for 10 min in 0.5% PBSD.</li>
		<li>Add the desired secondary antibody (488 donkey anti-rabbit for green fluorescence) at a dilution of 1:1000 in 0.5% PBSD for 16 h.</li>
		<li>Wrap the 24-well plate in aluminum and place at 4 &#176;C.</li>
		<li>Wash slices three times for 5 minutes in 0.5% PBSD.</li>
		<li>Wash for 5 min in PBS.</li>
		<li>Transfer slices with a pencil brush into a water container.</li>
		<li>Mount slices floating in the water on charged slides.</li>
		<li>Coverslip sections with hard-set mounting media Dopamine beta-hydroxylase</li>
		<li>Dry the mounted brain sections for 30 min at room temperature.</li>
		<li>Image brain slices at a confocal or fluorescent microscope with settings to detect signals from appropriate secondary antibody fluorophore wavelength.</li>
		<li>Adjust the microscope to the focal plane of the brain slice and take a single image at 10X magnification.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>ASIN: B000CMFJZ2</td>
			<td></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>Wide-field fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Axio Zoom.V16</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunohistochemistry for identifying the locus coeruleus region in mouse brain sections

Source: Schmidt, K. et al., <a href="https://app.jove.com/v/58652/localization-of-the-locus-coeruleus-in-the-mouse-brain">Localization of the Locus Coeruleus in the Mouse Brain.</a> J. Vis. Exp. (2019)
This protocol outlines the use of immunohistochemistry to identify locus coeruleus regions in mouse brain sections. By targeting dopamine &#946;-hydroxylase with green fluorophore-labeled antibodies, norepinephrine-producing neurons are visualized as green fluorescence under a microscope.

<img alt="Figure 1" src="/files/ftp_upload/23122/23122_Figure1.jpg" /> 
Figure 1: Detection of LC by immunostaining brain slices for DBH or TH. (a) One mouse brain was sectioned into 50 &#956;m slices around the brain stem and collected in two 24-well dishes. Every 5th to 8th collected brain slice was mounted on film covered coverslip for imaging via XFM. These slices are labeled with blue numbers between the wells. Adjacent slices floating in PBS were immunostained ...

Immunohistochemistry for Identifying the Locus Coeruleus Region in Mouse Brain Sections

Source: Schmidt, K. et al., Localization of the Locus Coeruleus in the Mouse Brain. J. Vis. Exp. (2019)
This protocol outlines the use of ...

The locus coeruleus regions, located bilaterally in the mouse brainstem, contain norepinephrine-producing neurons and regulate various regions across the brain.
To detect these locus coeruleus regions, begin with a glass slide carrying the fixed, cryopreserved mouse brainstem sections.
Wash the slide with a buffer to remove fixative traces.
Add a buffer containing detergent and incubate to permeabilize the membranes, then wash.
Introduce primary antibodies specific to dopamine &#946;-hydroxylase enzyme, which converts dopamine to norepinephrine, a neurotransmitter.
Incubate to allow the antibodies to interact with dopamine &#946;-hydroxylase.
Wash to remove unbound antibodies.
Then, add green fluorophore-coupled secondary antibodies and incubate in the dark.
The secondary antibodies bind to the primary antibodies and then wash.
Add mounting medium to the brain sections, then cover and dry.
Observe the slides under a fluorescence microscope.
The green fluorescence representing the labeled norepinephrine-producing neurons in the brain sections indicates the presence of locus coeruleus regions.

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20 Görüntüleme. Source: Schmidt, K. et al., Localization of the Locus Coeruleus in the Mouse Brain. J. Vis. Exp. (2019) This protocol outlines the use of immunohistochemistry to identify locus coeruleus regions in mouse brain sections. By targeting dopamine β-hydroxylase with green fluorophore-labeled antibodies, norepinephrine-producing neurons are visualized as green fluorescence under a microscope. 

Video: Immunohistochemistry for Identifying the Locus Coeruleus Region in Mouse Brain Sections - Kavram

Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive performance. These effects may be due to widespread connections of the trigeminal system to the ascending reticular activating system (ARAS), to which noradrenergic neurons of the locus coeruleus (LC) belongs. LC neurons contain projections to the whole brain, and it is known that their discharge co-varies with pupil size. LC activation is necessary for eliciting task-related mydriasis. If chewing effects on cognitive performance are mediated by the LC, it is reasonable to expect that changes in cognitive performance are correlated to changes in task-related mydriasis. Two novel protocols are presented here to verify this hypothesis and document that chewing effects are not attributable to aspecific motor activation. In both protocols, performance and pupil size changes observed during specific tasks are recorded before, soon after, and half an hour following a 2 min period of either: a) no activity, b) rhythmic, bilateral handgrip, c) bilateral chewing of soft pellet, and d) bilateral chewing of hard pellet. The first protocol measures level of performance in spotting target numbers displayed within numeric matrices. Since pupil size recordings are recorded by an appropriate pupillometer that impedes vision to ensure constant illumination levels, task-related mydriasis is evaluated during a haptic task. Results from this protocol reveal that 1) chewing-induced changes in performance and task-related mydriasis are correlated and 2) neither performance nor mydriasis are enhanced by handgrip. In the second protocol, use of a wearable pupillometer allows measurement of pupil size changes and performance during the same task, thus allowing even stronger evidence to be obtained regarding LC involvement in the trigeminal effects on cognitive activity. Both protocols have been run in the historical office of Prof. Giuseppe Moruzzi, the discoverer of ARAS, at the University of Pisa.

To verify whether trigeminal effects on cognitive performance involve locus coeruleus activity, two protocols are presented that aim to evaluate possible correlations between the performance and task-related pupil size changes induced by chewing. These protocols may be applied to conditions in which locus coeruleus contribution is suspected.

Trigeminal Stimülasyon tarafından Ortaya Çıkan Locus Coeruleus aracılı Uyarılma'da Gözbebeği Ile Bağlantılı Değişikliklerin Değerlendirilmesi

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive ...

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Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Multipleks Floresan In Situ Hibridizasyonun Floresan İmmünohistokimya ile Taze Dondurulmuş veya Sabit Fare Beyin Kesitlerinde Birleştirilmesi

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Detection of Abnormal Prion Protein by Immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

Immunohistochemistry for Identifying the Locus Coeruleus Region in Mouse Brain Sections

TRANSKRIPT

Immunohistochemistry for Identifying the Locus Coeruleus Region in Mouse Brain Sections

Trigeminal Stimülasyon tarafından Ortaya Çıkan Locus Coeruleus aracılı Uyarılma'da Gözbebeği Ile Bağlantılı Değişikliklerin Değerlendirilmesi

Multipleks Floresan In Situ Hibridizasyonun Floresan İmmünohistokimya ile Taze Dondurulmuş veya Sabit Fare Beyin Kesitlerinde Birleştirilmesi

İmmünohistokimya ile Anormal Prion Proteininin Saptanması