The authors would like to thank Hartwig Wieboldt for his excellent technical assistance, and the UKE Microscopy Imaging Facility (Umif) of the University Medical Center Hamburg-Eppendorf for providing microscopes and support. This research was funded by the DZHK (German Centre for Cardiovascular Research) [FKZ 81Z4710141].

All procedures involving animals were approved by the Animal Care and Use Committee of the State of Hamburg (ORG870, 959) and the North Rhine-Westphalian State Agency for Nature, Environment and Consumer Protection (LANUV, 07/11) and conform to the National Institutes of Health&#39;s Guide for the Care and Use of Laboratory Animals (2011). Studies were performed using male and female (aged 10-24 weeks) C57BL/6 mice (stock number 000664, Jackson Laboratories) and mice homozygous (db/db) or heterozygous (db/het; control) for the diabetes spontaneous mutation (Leprdb; BKS.Cg-Dock7m+/+ Leprdb&#160;/J, stock number 000642, Jackson Laboratories). The authors have used the protocols at hand without variations for mice aged up to 60 weeks.
1. Location and dissection of murine stellate ganglia
NOTE: Even though descriptions and drawings are mostly available in bigger species, some publications have previously described the location of the SG in rats24 and mice25 using anatomical methods and fluorescent reporter lines, respectively.
<ol>
	<li>Prepare 50 mL of ice-cold (3-4 &#176;C) heparinized (20&#160;units/mL, see Table of Materials) phosphate-buffered saline (PBS). Perform dissection of the SG at room temperature (RT).</li>
	<li>Deeply anesthetize mouse by inhalation of 3%-5% isoflurane according to the institutional and local guidelines. Verify adequate anesthesia by loss of pedal withdrawal reflex. Decapitate mice or perform cervical dislocation. 
	NOTE: Incorrect cervical dislocation can result in breakage of the spine and damage of thoracic vessels leading to bleeding which hinders the preparation or&#160;severing of the sympathetic chain, so that SG are not in their correct position. Therefore, it is critical to have experienced personnel perform cervical dislocation or decapitate animals in deep sedation.</li>
	<li>Spray the skin with ethanol and open the thorax with two incisions along the anterior axillary lines using Mayo scissors and narrow pattern forceps. Cut the diaphragm and remove the front of the&#160;ribcage.</li>
	<li>Remove the heart-lung package by gripping the aorta and vena cava right above the diaphragm using London forceps and cutting all vessels and connective tissue close to the spine below with the Strabismus scissors.</li>
	<li>Flush the thorax thoroughly with heparinized PBS using a plastic disposable pipette until all traces of blood are removed.</li>
	<li>Place the torso under stereomicroscope preparation binoculars and ensure good lighting in the thorax with external light sources.</li>
	<li>Locate the first rib and the longus colli muscles. 
	NOTE: The SG are located bilaterally, parallel to the spine at the branch between the first rib and the spine, in a groove lateral to the longus colli muscles24. The flat side of the SG is located adjacent to the longus colli muscles. Depending on the preparation, parts of the sympathetic chain might already be visible as white, oblique fibers parallel to the spine. These can be traced along to the SG.</li>
	<li>Gently use the tip of the Dumont #5/45 forceps to expose the connective tissue lateral to the longus colli muscle.</li>
	<li>Turn the forceps around by 180&#176; and use the flat side to grip the SG and pull it out with minimal pressure.</li>
	<li>Repeat with the second SG.</li>
	<li>Place both SG in a dish (6 cm diameter) filled with cold PBS and inspect with the appropriate magnification. If necessary, remove excess vessels, fat tissue, and larger nerves. 
	NOTE: Autonomic ganglia are surrounded by a connective tissue capsule consisting of collagen fibers and fibroblasts26,27.The permeability of these capsules seems to vary among species, different kind of ganglia26 and age28. Remove as much connective tissue as possible using Dumont #5/45 forceps and, if necessary, spring scissors.</li>
	<li>Depending on the goal of the experiment, proceed with section 2, 3, or 5&#160;of this protocol.</li>
</ol>
2. Whole mount immunohistochemistry protocol
NOTE: This protocol is adapted from cardiac whole mount stainings4,29. Perform incubation steps for every single SG in one well of a 96-well plate and use 100 &#956;L (for antibody-containing solutions) to 200 &#956;L (for all other solutions) of the solution to ensure complete coverage. Regularly check the coverage and correct immersion of the SG with binoculars. Remove liquids manually with a 200 &#956;L pipette with an additional 10 &#956;L tip on top of the 200 &#956;L tip. This will prevent aspiration of the SG in the pipette tip. Use freshly prepared solutions and sterile liquids to prevent bacterial growth.
<ol>
	<li>Fix SG for histology for 2 h at RT in 4% methanol-free paraformaldehyde (PFA)/PBS.</li>
	<li>Process SG as quickly as possible, but they can be stored for 2-4 weeks at 4-6 &#176;C in PBS with 0.02% (w/v) sodium azide at this point.</li>
	<li>Prepare Sudan black stock solution (1% Sudan black w/v in 100% ethanol) for reduction of autofluorescence and improvement of signal to background ratio30. Dissolve for 2-3 h on a magnetic stirrer at RT. 
	NOTE: Use the stock solution for a maximum of 6-8 weeks, discard earlier when sedimentation appears.</li>
	<li>Prepare Sudan black working solution by centrifuging the stock solution for 30 min at full speed (13,000 x g) to remove debris and diluting the stock in 70% ethanol to a final concentration of 0.25% Sudan black.</li>
	<li>Treat SG with Dent&#39;s bleach to improve antibody permeabilization31. Freshly prepare Dent&#39;s bleach by mixing methanol (MeOH), hydrogen peroxide solution 30% (w/w) in H2O and dimethyl sulfoxide (DMSO) in a ratio of 4:1:1. Add 200 &#956;L per SG and place the plate on an orbital shaker for 1 h at RT.</li>
	<li>Perform a descending MeOH series for rehydration by incubating for 10 min each on an orbital shaker: 100% MeOH, 75% MeOH/PBS, 50% MeOH/PBS, 25% MeOH/PBS.</li>
	<li>Perform permeabilization by incubating SG twice for 60 min each in PBS/1% Triton-X-100 at RT.</li>
	<li>Remove permeabilization solution from the SG and add Sudan black working solution. Incubate for 2 h at RT on an orbital shaker.</li>
	<li>In the meantime, prepare a blocking solution by adding 5% receptor grade bovine serum albumin (BSA) and 0.1% Triton-X-100 in PBS a 15 mL vessel and let it dissolve on a roller shaker for approximately 5-10 min. Decant through a pre-pleated paper filter to remove debris.</li>
	<li>Remove Sudan black very carefully by tilting the plate and carefully pipetting from the upright side. 
	NOTE: It is not possible to see the SG in Sudan black. Use a strong light source and work slowly. From this step on, SG are stained black, enhancing visibility. If any additional connective tissue is seen surrounding SG at this point, remove it using Dumont #5/45 forceps and spring scissors.</li>
	<li>Add 200 &#956;L of PBS/0.1% Triton-X-100 (PBS-T) and wash for 5 min at RT on an orbital shaker.</li>
	<li>Remove PBS-T by aspirating it with a pipette and repeat 2 times.</li>
	<li>Remove PBS; add 200 &#956;L of blocking solution and incubate at 4 &#176;C overnight on an orbital shaker.</li>
	<li>On the next day, prepare solutions by adding primary antibodies in the blocking solution. Adapt antibody concentrations from established protocols. 
	NOTE: Include one SG as antibody control without primary antibody (incubated with antigen-preabsorbed antibody or IgG, if available, or blocking buffer).</li>
	<li>Perform primary antibody incubation for 36-48 h at 4 &#176;C on an orbital shaker. For cell size measurements and staining of sympathetic neurons, use antibodies against tyrosine hydroxylase (see Table of Materials for antibody recommendations). 
	NOTE: Place the 96-well plate in a wet chamber (e.g., plastic box lined with ddH2O-wetted paper towels) to prevent evaporation at this point.</li>
	<li>Remove antibody solution carefully and add 200 &#956;L of PBS-T. Place the plate on an orbital shaker for 30 min.</li>
	<li>Remove PBS-T and repeat the washing step 5 additional times.</li>
	<li>Prepare the secondary antibody working solution by centrifuging fluorescent Alexa-labeled secondary antibodies for 1 min at full speed (13,000 x g) before usage. Dilute the appropriate secondary antibodies according to the primary antibodies 1:500 in the blocking solution and add to SG. Add 1 &#956;g/mL of bisbenzimide H33342 trihydrochloride (Hoechst staining) if nuclear staining is desired. Incubate for 12-24 h at 4 &#176;C on an orbital shaker.</li>
	<li>Remove the antibody solution carefully and add 200 &#956;L of PBS-T. Place the plate on an orbital shaker for 30 min.</li>
	<li>Remove PBS-T and repeat the washing step 5 additional times. For the last step, use PBS without Triton.</li>
	<li>For embedding, spread 50-100 &#956;L of fluorescent mounting medium (see Table of Materials) on a glass slide and place under preparation binoculars. Use Dumont #5/45 forceps to pick up SG from the 96-well plate and remove excess liquid by dipping one end on a filter paper (e.g., Whatman drying pad) and place it on the drop.</li>
	<li>Use Dumont #5/45 forceps to correct positioning with the appropriate magnification.</li>
	<li>Gently place a glass coverslip (20 mm x 20 mm) next to the SG and slowly descend. 
	NOTE: Using too much mounting medium will result in movement of the SG or tangling of nerves. If that happens, quickly remove the coverslip and repeat steps 2.9-2.21.</li>
	<li>Let the slides dry in the dark overnight at RT. Storage of the stained specimen is possible for at least 4-6 weeks at 4 &#176;C.</li>
</ol>
3. Whole mount in situ hybridization
NOTE: Whole-mount in situ-hybridization of the SG is adapted from the organ of corti32 and the commercial RNA fluorescence in situ protocol (see Table of Materials). Obtain probes for the genes of interest and buffers and solutions from the supplier. All incubation steps are performed at RT, if not mentioned otherwise. Use sterile PBS. If interested in staining several SG in one well, use at least 150 &#956;L of buffers and solutions.
<ol>
	<li>Perform dissection of the SG as described in steps 1.1-1.13.</li>
	<li>Fixate SG for 1 h in 200 &#956;L of 4% MeOH-free PFA/PBS in one well of a 96-well plate placed on an orbital shaker.</li>
	<li>Wash SG three times for 30 min each in 0.1% Tween-20/PBS on an orbital shaker.</li>
	<li>Dehydrate SG in MeOH/PBS series by subsequent incubation in 50% MeOH/PBS, 70% MeOH/PBS, and 100% MeOH for 10 min each on an orbital shaker.</li>
	<li>Store SG at -20 &#176;C in 100% MeOH overnight.</li>
	<li>The next day, pre-warm the incubator to 40 &#176;C. Check the temperature with a thermometer.</li>
	<li>Rehydrate SG in reverse MeOH/PBS series (100% MeOH, 70% MeOH/PBS, 50% MeOH/PBS) for 10 min each.</li>
	<li>Wash SG three times for 5 min each in PBS.</li>
	<li>In the meantime, start pre-warming the probes by incubation at 40 &#176;C for 10 min, followed by cooling for 10 min.</li>
	<li>Incubate SG in 200 &#956;L of Protease III for 15 min.</li>
	<li>Optional: Perform Sudan black treatment to quench autofluorescence according to sections 2.3, 2.4, and 2.8 of this protocol if subsequent immunofluorescence staining is planned.</li>
	<li>Wash SG in 200 &#956;L of 0.1% Tween-20/PBS 3x for 5 min each on an orbital shaker.</li>
	<li>Optional: If co-staining with several probes is desired, dilute Channel-2 (50x) and Channel-3 probe (50x) in Channel-1 probe (1x).</li>
	<li>Cover SG with 100 &#956;L of probe for the gene of interest and incubate overnight at 40 &#176;C with slight agitation. Place the 96-well plate in a wet chamber at 40 &#176;C for all incubation steps. 
	NOTE: Include one SG as negative control, using a probe against a bacterial gene (e.g., dihydro-dipicolinate reductase, Dapb) to check for non-specific binding of amplification reagents in later steps.</li>
	<li>Wash SG in supplied washing buffer 3x for 15 min each on an orbital shaker.</li>
	<li>Pre-warm Amp1-3, HRP-C1, and HRP-Blocker to RT. If co-staining with Channel-2 and/or Channel-2 probe is desired, pre-warm HRP-C2 and HRP-C3.</li>
	<li>Re-fix SG for 10 min at RT in 4% PFA/PBS on an orbital shaker.</li>
	<li>Wash SG in 200 &#956;L of supplied washing buffer 3x for 5 min each on an orbital shaker at RT.</li>
	<li>For amplification, incubate SG with 100 &#956;L of Amp1 for 35 min at 40 &#176;C on an orbital shaker.</li>
	<li>Carefully remove any liquid and wash SG in 200 &#956;L of supplied washing buffer 3x for 5 min each at RT on an orbital shaker.</li>
	<li>Incubate SG with 100 &#956;L of Amp2 for 35 min at 40 &#176;C on an orbital shaker.</li>
	<li>Repeat step 3.18.</li>
	<li>Incubate SG with 100 &#956;L of Amp3 for 20 min at 40 &#176;C on an orbital shaker.</li>
	<li>Repeat step 3.18.</li>
	<li>Incubate SG with 100 &#956;L of supplied Multiplex FL v2 HRP-C1 for 20 min at 40 &#176;C on an orbital shaker.</li>
	<li>Repeat step 3.18.</li>
	<li>Prepare Opal-conjugated secondary antibody 1:1,000 in 200 &#956;L of supplied TSA-buffer and incubate SG for 35 min at 40 &#176;C on an orbital shaker. Protect from light during incubation and from this step on.</li>
	<li>Repeat step 3.18.</li>
	<li>Incubate SG in 100 &#956;L of supplied Multiplex FL v2 HRP-blocker for 15 min at 40 &#176;C on an orbital shaker.</li>
	<li>Repeat step 3.18.</li>
	<li>Optional: For co-staining with Channel-2 probe, repeat steps 3.25-3.30 with supplied Multiplex FL v2 HRP-C2.</li>
	<li>Optional: For co-staining with Channel-3 probe, repeat steps 3.25-3.30 with supplied Multiplex FL v2 HRP-C3.</li>
	<li>In case of subsequent immunofluorescent staining, perform steps 2.13-2.25 of this protocol.</li>
	<li>Incubate in 1% BSA/PBS for 30 min on an orbital shaker.</li>
	<li>Incubate SG for 30 min in 1 &#956;g/mL of bisbenzimide H33342 trihydrochloride (Hoechst staining) in 1% BSA/PBS if nuclear staining is desired and/or add Alexa-coupled wheat germ agglutinin (WGA, 1:500) on an orbital shaker.</li>
	<li>Repeat step 3.18.</li>
	<li>Embed as described in steps 2.19-2.22.</li>
</ol>
4. Imaging and analyses of murine stellate ganglia
<ol>
	<li>Perform confocal microscopy of embedded SG in the local imaging facility.</li>
	<li>If cell size measurements are required, image SG stained for tyrosine hydroxylase (see Table of Materials) at 200x magnification and take 4-6 random images from every SG.</li>
	<li>Analyze images using ImageJ33 software to estimate cell size (e.g., with a pen table, see Table of Materials). Use Free Hand Selection, circle each cell and click on Analyze | Measure to obtain cell area. Be careful to include only intact, fully visible cells that are located well within the SG.</li>
	<li>Using this method, perform measurement of approximately 100 cells per SG.</li>
	<li>Have a blinded investigator perform steps 4.2-4.3 if you want to compare SG from different mice. Use a frequency distribution in statistical software to visualize size differences between groups34.</li>
</ol>
5. Molecular analyses of murine stellate ganglia
NOTE: Include controls depending on your experimental design. This could be SG with different genotypes and disease background and/or other autonomic ganglia, such as the sympathetic superior cervical ganglion (located in the neck area, see detailed description in Ziegler et al.35) or parasympathetic ganglia (such as intracardiac ganglia, see Jungen et al.4).
<ol>
	<li>Prepare a 2 mL tube with 500 &#956;L of phenol/guanidine thiocyanate solution (e.g., Qiazol) per animal and have liquid nitrogen ready for shock-frosting or consider commercial solutions for protection of RNA (optional, see Table of Materials)36. Work quickly for RNA isolation.</li>
	<li>Perform dissection of the SG as described in steps 1.1-1.13.</li>
	<li>Immediately immerse both SG directly in one tube with phenol/guanidine thiocyanate solution and shock-frost tube in liquid nitrogen.</li>
	<li>Store at -80 &#176;C until further processing.</li>
	<li>For tissue lysis, let tubes with SG thaw until phenol/guanidine thiocyanate solution is liquified and add two 7 mm stainless steel beads. Cool down the metal parts of tissue homogenizer (ball or mixer mill, e.g., Tissue Lyser II) on dry ice and centrifuge at&#160;4 &#176;C.</li>
	<li>Centrifuge tubes at 500 x g for 1 min at 4 &#176;C so that SG are at the bottom of the tube.</li>
	<li>Put tubes into the metal parts of the Tissue Lyser and lyse for 1 min at 20 Hz.</li>
	<li>Repeat steps 5.6 and 5.7 up to 5 times until no intact tissue is detectable.</li>
	<li>Transfer the liquid into a fresh 1.5 mL tube.</li>
	<li>Perform RNA isolation with a column-based RNA isolation kit (e.g., miRNeasy mini kit) according to the manufacturer&#39;s instructions.</li>
	<li>Elute RNA in 20 &#956;L of RNase-free water and measure concentration using a spectrophotometer. 
	NOTE: To exclude contamination of the purified RNA with genomic DNA, we propose performing a polymerase chain reaction with genomic primers and 1 &#956;L of RNA as template, instead minus reverse transcriptase control. This will save a significant amount of RNA. If RNA is contaminated, use exon-intron boundary primers or intron flanking primers for subsequent quantitative real-time polymerase chain reaction.</li>
	<li>Use 250 ng SG RNA to perform cDNA synthesis and use established protocols. Here, a high-capacity cDNA reverse transcription kit was used according to the manufacturer&#39;s instructions.</li>
	<li>Dilute to a final concentration of 2.5 ng/&#956;L of cDNA and perform quantitative real-time polymerase chain reaction with the appropriate probes according to the established protocols. Here, TaqMan Assay (see Table of Materials) was performed using 10 ng cDNA per reaction. 
	NOTE: Perform no-template control for every gene to exclude false positive results.</li>
	<li>Normalize gene expression of your gene of interest on a house keeping gene (e.g., Cdkn1b) to compare relative gene expression between different groups of SG.</li>
</ol>

The authors have nothing to disclose.

The understanding of cellular and molecular processes in neurons and glial cells of the sympathetic nervous system that precede the onset of VA is of high interest, as sudden cardiac arrest remains the most common cause of death worldwide5. Therefore, in the current manuscript, we provide a basic repertoire of methods to identify the murine SG&#160;&#8211;&#160;a murine&#160;element within&#160;this network &#8211;&#160;and perform subsequent analyses on RNA, protein, and cellular level.
<p cl...

The cardiac autonomic nervous system is a tightly regulated equilibrium of sympathetic, parasympathetic, and sensory components that allows the heart to adapt to environmental changes with the appropriate physiological response1,2. Disturbances in this equilibrium, for example, an increase of sympathetic activity, have been established as a key driver for the onset as well as maintenance of ventricular arrhythmias (VA)3,4. Therefore, autonomic modulation, achieved via pharmacological reduction of sympathetic activity with beta-blockers, has been a cornerstone in the treatment of patients with VA for decades5,6. But despite pharmacological and catheter-based interventions, a relevant number of patients still suffers from recurrent VA7.
Sympathetic input to the heart is mostly mediated via neuronal cell bodies in the stellate ganglia (SG), bilateral star-shaped structures of the sympathetic chain, which relay information via numerous intrathoracic nerves from the brainstem to the heart8,9,10. Nerve sprouting from the SG after injury is associated with VA and sudden cardiac death11,12, emphasizing the SG as a target for autonomic modulation13,14. A reduction of sympathetic input to the heart can be achieved temporarily via percutaneous injection of local anesthetics or permanently by partial removal of the SG via video-assisted thoracoscopy15,16. Cardiac sympathetic denervation presents an option for patients with therapy-refractory VA with promising results14,16,17. We have learned from explanted SG of these patients that neuronal and neurochemical remodeling, neuro-inflammation and glial activation are hallmarks of sympathetic remodeling that might contribute or aggravate autonomic dysfunction18,19. Still, the underlying cellular and molecular processes in these neurons remain obscure to date, for example, the role of neuronal transdifferentiation into a cholinergic phenotype20,21. Experimental studies present novel approaches to treat VA, for example, the reduction of sympathetic nerve activity via optogenetics22, but in-depth characterization of the SG is still lacking in many cardiac pathologies that go in hand with VA. Mouse models mimicking these pathologies allow to study neuronal remodeling that potentially precedes the onset of arrhythmias12,23. These can be completed by further morphological and functional analyses for autonomic characterization of the heart and the nervous system. In the present protocol, we provide a basic repertoire of methods allowing to dissect and characterize the murine SG to improve the understanding of VA.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plate</td>
			<td>TPP</td>
			<td>92097</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Adhesion Slides SuperFrost plus&#160; 25 x 75 x 1 mm</td>
			<td>R. Langenbrinck</td>
			<td>03-0060</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Albumin bovine Fraction V receptor grade lyophil.</td>
			<td>Serva</td>
			<td>11924.03</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>bisBenzimide H33342 trihydrochloride (Hoechst)</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>B2261</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Chicken anti neurofilament</td>
			<td>EMD Millipore</td>
			<td>AB5539</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>D8418</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey anti chicken IgY Alexa 647&#160;</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>AP194SA6</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey anti goat IgG Alexa 568&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11057</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey anti rabbit IgG Alexa 488&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Drying block 37-100 mm</td>
			<td>Whatman (Sigma Aldrich)</td>
			<td>WHA10310992&#160;</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma Aldrich</td>
			<td>E4009</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Ethanol 99 % denatured with MEK, IPA and Bitrex (min. 99,8 %)</td>
			<td>Th.Geyer</td>
			<td>2212.5000</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Eukitt mounting medium</td>
			<td>AppliChem</td>
			<td>253681.0008</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Fluoromount-G + DAPI</td>
			<td>Southern Biotech</td>
			<td>0100-20</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Goat anti choline acetyltransferase</td>
			<td>EMD Millipore</td>
			<td>AP144P</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>H2O2 30% (w/w)</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>H1009</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Heparin Sodium 25.000 UI / 5ml</td>
			<td>Rotexmedica</td>
			<td>PZN: 3862340</td>
			<td>Preparation SG</td>
		</tr>
		<tr>
			<td>High-capacity cDNA reverse transctiption kit</td>
			<td>Life technologies</td>
			<td>&#160;4368813</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Isoflurane (Forene)</td>
			<td>Abbott Laboratories</td>
			<td>2594.00.00</td>
			<td>Preparation SG</td>
		</tr>
		<tr>
			<td>Mayer&#39;s hemalum solution</td>
			<td>Merck</td>
			<td>1.09249.0500</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Microscope cover glasses 20x20 mm or smaller</td>
			<td>Marienfeld</td>
			<td>0101040</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>miRNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>NanoDrop 2000c</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000C</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Opal 570 Reagent Pack</td>
			<td>Akoya Bioscience</td>
			<td>FP1488001KT</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16% w/v aq. soln., methanol free&#160;</td>
			<td>Alfa Aesar</td>
			<td>43368</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Pasteur pipettes, LDPE, unsterile, 3 ml, 154 mm</td>
			<td>Th.Geyer</td>
			<td>7691202</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline tablets</td>
			<td>Gibco</td>
			<td>18912-014</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Pinzette Dumont SS Forceps</td>
			<td>FineScienceTools</td>
			<td>11203-25</td>
			<td>Preparation SG</td>
		</tr>
		<tr>
			<td>QIAzol Lysis Reagent</td>
			<td>Qiagen&#160;</td>
			<td>79306</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Rabbit anti tyrosine hydroxylase</td>
			<td>EMD Millipore</td>
			<td>AB152</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Merck</td>
			<td>R0901-100ML</td>
			<td>RNA isolation (optional)</td>
		</tr>
		<tr>
			<td>RNAscope Multiplex Fluorescent Reagent Kit v2</td>
			<td>biotechne (ACD)</td>
			<td>323100</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>RNAscope Probe-Mm-S100b-C2</td>
			<td>biotechne (ACD)</td>
			<td>431738-C2</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>RNAscope Probe-Mm-Tubb3</td>
			<td>biotechne (ACD)</td>
			<td>423391</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Stainless steel beads 7 mm&#160;</td>
			<td>Qiagen&#160;</td>
			<td>69990</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Sudan black B</td>
			<td>Roth</td>
			<td>0292.2</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Cdkn1b (Mm00438168_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Choline acetyltransferase (Mm01221880_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay MKi67 (Mm01278617_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay PTPCR (Mm01293577_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay S100b (Mm00485897_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Tyrosin Hydroxylase (Mm00447557_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan mastermix</td>
			<td>Applied biosystems</td>
			<td>4370074</td>
			<td>Gene Expression analysis&#160;</td>
		</tr>
		<tr>
			<td>Tissue Lyser II</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Triton X-100 10% solution</td>
			<td>Sigma-Aldrich</td>
			<td>93443-100ml</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-100ML</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Wacom bamboo pen</td>
			<td>Wacom</td>
			<td>CTL-460/K</td>
			<td>Cell size measurements</td>
		</tr>
		<tr>
			<td>Whatman prepleated qualitative filter paper, Grade 595 1/2</td>
			<td>Sigma-Aldrich</td>
			<td>WHA10311647</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>W21404</td>
			<td>RNAscope</td>
		</tr>
	</tbody>
</table>

location, dissection, and analysis of the murine stellate ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Figure 1&#160;visualizes how to identify and dissect the SG. Figure 1A shows a schematic drawing of the location, while Figure 1B presents the view into&#160;the thorax after removal of the heart-lung-package. The left and right longus colli muscles medial from the SG and the rib cage are important landmarks for orientation. Dissection is performed along the dotted lines between muscles and the first rib. The SG and the sympathetic ...

Watch this Scientific Journal Video about Location, Dissection, and Analysis of the Murine Stellate Ganglion at JoVE.com

Location, Dissection, and Analysis of the Murine Stellate Ganglion

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

location-dissection-and-analysis-of-the-murine-stellate-ganglion

Research

JoVE Journal

Medicine

9.5K Views.  EVK Düsseldorf, cNEP, cardiac Neuro- and Electrophysiology Research Consortium. Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

Video: Location, Dissection, and Analysis of the Murine Stellate Ganglion

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &amp;#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Robotic Left Hepatectomy using Indocyanine Green Fluorescence Imaging for an Intrahepatic Complex Biliary Cyst

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. Surgery is the cornerstone treatment for patients with BC. While total BC excision and Roux-Y hepaticojejunostomy is the treatment method of the choice in patients with extrahepatic BC (i.e., Todani I-IV), patients with intrahepatic BC (i.e., Todani V) benefit the most from a surgical liver resection. In recent years, minimally invasive liver surgery (MILS) including robotic MILS has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. Robotic major MILS is still considered technically demanding and a detailed description of the technical approach during robotic major MILS has only been limitedly discussed in the literature. The current article describes the main steps for a robotic left hepatectomy in a patient with a large BC Todani Type V. The patient is in French position with 5 trocars placed (4 robotic, 1 laparoscopic assistant). After mobilizing the left hemiliver, the left and right hepatic artery are dissected carefully followed by a cholecystectomy. Intraoperative ultrasound is performed to confirm localization and margins of the BC. The Left hepatic artery and left portal vein are isolated, clipped, and divided. Indocyanine green (ICG) fluorescence imaging is used regularly during the entire procedure to visualize and confirm biliary tract anatomy and the BC. Parenchymal transection is performed with robotic cautery hook for the superficial part and robotic cautery spatula, bipolar cautery, and vessel sealer for the deeper parenchyma. The postoperative course was uncomplicated. A robotic left hepatectomy is technically demanding, yet a feasible and safe procedure. ICG-fluorescence imaging aids in delineating the BC and bile duct anatomy. Further, comparative studies are needed to confirm clinical benefits of robotic MILS for benign and malignant indications.

Robotic liver surgery has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. However, robotic left hepatectomy is still technically demanding. We describe our surgical technique of a robotic left hepatectomy using indocyanine green fluorescence imaging for a large biliary cyst.

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. ...