Name: dissection of human vitreous body elements for proteomic analysis
Uploaded: 2011-01-23T17:45:00
Duration: 5 min 5 s
Description: Watch this Scientific Journal Video about Dissection of Human Vitreous Body Elements for Proteomic Analysis at JoVE.com

Funding was provided by Fight for Sight. Tissues were obtained from the Iowa Lions Eye Bank.

1. Anterior Segment Dissection.
<ol>
<li>The cornea is removed by first making an incision at the limbus into the anterior chamber using a 15&deg; blade (Figure 1). Then one blade of curved cornea-scleral scissors is inserted into the anterior chamber. Circumferential cuts are made in the cornea, just anterior to the limbus.</li>
<li>0.12 Colibri forceps and Westcott scissors are used to cut the iris circumferentially anterior to the ciliary body.</li>
<li>The lens nucleus is removed with 0.12 Colibri forceps or a 15&deg; blade (supersharp) and the cortex and capsule with forceps. </li>
</ol>
2. Vitreous Core Aspiration.
<ol>
<li>A 23-gauge needle on a 5-cc syringe is inserted into the mid-vitreous (Figure 1).</li>
<li>Approximately 1 mL or more of vitreous core is gently aspirated.</li>
<li>The sample is then placed in a microfuge tube and then into liquid nitrogen.</li>
</ol>
3. Anterior Hyaloid Dissection.
<ol>
<li>The anterior hyaloid is seen as a semi-transparent ring by adjusting the incident light from a goose neck microscope light. Previous aspiration of the vitreous core separates the anterior hyaloid from other structures for easier identification.</li>
<li>The sheet of elastic tissue is carefully pulled away from the ciliary body with Colibri or dressing forceps and cut with Vannas scissors. This maneuver is repeated.</li>
<li>The sample is then placed in a microfuge tube and then into liquid nitrogen.</li>
</ol>
4. Vitreous Base Dissection.
<ol>
<li>Using 0.12 forceps to stabilize the eye cup, Westcott scissors are used to "flower" the eye by making four equally spaced stress-relieving cuts from the ciliary body towards the optic nerve head.</li>
<li>The pars plicata of the ciliary body (Figure 2) is removed from each of the quadrants using Westcott scissors.</li>
<li>The vitreous base is then grasped on either side of the ora serrata with forceps over the pars plana and the retina (Figure 2).</li>
<li>Continuous traction on the vitreous base is applied with the forceps. Sequential cutting with Westcott scissors extracts a semi-transparent tissue with a "string of pearls" appearance as it is excised.</li>
<li>The sample is then placed in a microfuge tube and then into liquid nitrogen.</li>
</ol>
5. Vitreous Cortex Removal.
<ol>
<li>The pars plana is excised from each of the quadrants with Westcott scissors by cutting the leaflet approximately 3-mm posterior to the ora serrata (Figure 2).</li>
<li>Between the tissue leaflets, the vitreous cortex is visualized as a semi transparent film.</li>
<li>The elastic film is either grasped with 0.12 forceps or held by adherence to a Weck-Cel surgical sponge. The vitreous cortex is then pulled away from the retina and excised with Westcott scissors (Figure 1).</li>
<li>The sample is placed in a microfuge tube and then into liquid nitrogen. All samples are stored at -80&deg; C until utilized for experimentation.</li>
</ol>
6. Representative Results
Tissue samples can be processed by a variety of methods for specific experiments. In our case, samples were submitted for protein analysis by SDS-PAGE (Figure 3).
 <img src="/files/ftp_upload/2455/2455fig1.jpg" alt="Figure 1" /> 
Figure 1. Cross-sectional view of the human eye depicting different substructures of the vitreous body. The most anterior vitreous is a thin collagenous layer called the anterior hyaloid. The vitreous core comprises the entire central region of the vitreous body. This portion of the vitreous is more aqueous in contrast to the vitreous base, which is viscous enough to be grasped by forceps and is firmly attached to the underlying ciliary body and retina. Encompassing the vitreous core is a very thin collagenous shell called the vitreous cortex.
 <img src="/files/ftp_upload/2455/2455fig2.jpg" alt="Figure 2" /> 
Figure 2. Vitreous base anatomy. The vitreous base is a semi-transparent substructure of the vitreous body located along the ora serrata (white arrows), which is the dividing line separating the ciliary body and retina. The anterior border of the vitreous base extends over the pars plana (white line) of the ciliary body. The posterior border of the vitreous base extends 2-3-mm posterior to the ora serrata (white dash). To excise the vitreous base, forceps are used to grasp the tissue and pull it away from the underlying ciliary body and retina. Once elevated, Westcott scissors are used to cut along the base.
 <img src="/files/ftp_upload/2455/2455fig3.jpg" alt="Figure 3" /> 
Figure 3. One-dimensional SDS-PAGE of Vitreous Body Elements. Total protein concentrations for the anterior hyaloid, vitreous base, vitreous core, and vitreous cortex were 11.24, 20.1, 16.61, 14.24 mg/mL, respectively. Gel electrophoresis was performed at 200 kV for 45 minutes, stained with Flamingo (Bio-Rad), and visualized using a VersaDoc Imaging system (Bio-Rad). The profiles for the different tissues show several similar bands, indicating either conserved or cross-contaminating proteins, as well as unique bands (asterisk), indicating differentially localized proteins.

<ol>
	<li>Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+macromolecules+and+supramolecular+organisation+of+the+vitreous+gel.">Structural macromolecules and supramolecular organisation of the vitreous gel.</a> Prog Retin Eye Res. 19, 323-344 (2000).</li><li>Sebag, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pharmacologic+vitreolysis.">Molecular biology of pharmacologic vitreolysis.</a> Trans Am Ophthalmol Soc. 103, 473-494 (2005).</li><li>Yoshimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+analysis+of+inflammatory+immune+mediators+in+vitreoretinal+diseases.">Comprehensive analysis of inflammatory immune mediators in vitreoretinal diseases.</a> PLoS One. 4, e8158-e8158 (2009).</li><li>Gao, B. B., Chen, X., Timothy, N., Aiello, L. P., Feener, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+vitreous+proteome+in+diabetes+without+diabetic+retinopathy+and+diabetes+with+proliferative+diabetic+retinopathy.">Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy.</a> J Proteome Res. 7, 2516-2525 (2008).</li><li>Izuta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+SOD+and+VEGF+are+increased+in+vitreous+bodies+from+proliferative+diabetic+retinopathy+patients.">Extracellular SOD and VEGF are increased in vitreous bodies from proliferative diabetic retinopathy patients.</a> Mol Vis. 15, 2663-2672 (2009).</li><li>Azimzadeh, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formalin-fixed+paraffin-embedded+(FFPE)+proteome+analysis+using+gel-free+and+gel-based+proteomics.">Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics.</a> J Proteome Res. 9, 4710-4720 (2010).</li></ol>

No conflicts of interest declared.

The vitreous body is a semi-transparent gel whose molecular composition is poorly understood, especially at the level of its substructures: the vitreous base, core, cortex, and anterior hyaloid. The vitreous core contains collagens II, V, IX, and XI, along with chondroitin sulphate proteoglycans, heparan sulphate proteoglycans, and hyaluronan.1,2 Protein biomarkers in the vitreous core have been associated with diseases such as diabetic retinopathy.3-5 How these proteins are differentially expressed in each of the substructures, and in many cases the specific protein identities, are not known. These details may give insight to the origin of proteins associated with specific vitreoretinal diseases and help target future therapies. Although the optimum post mortem interval for tissue dissection has not been determined, protein degradation may affect downstream experiments. For example, immunohistochemistry is affected in 12-hour postmortem eyes and some specific enzyme activities may be reduced within a few hours (unpublished observation). All tissues in this study were collected between 2 and 8 hours of death without significant changes in protein expression or suitability for proteomics analsyis. The liquid nitrogen freezing method of preservation is chosen over fixation in order to prevent small changes in protein structure caused by fixative cross linking, which has been demonstrated in other tissues by LC-MS/MS.6 Proteomic studies will depend on the ability to accurately dissect the different compartments of the vitreous, as demonstrated in this video experiment. We have validated the dissection technique using 1-dimensional SDS-PAGE. As our results suggest, there are differentially expressed proteins in the various vitreous body substructures. Identifying these proteins will provide a more detailed understanding of vitreous compartmentalization.

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 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 </tr>
 <tr>
 <td>0.12 forceps</td>
 <td>Storz Ophthalmics</td>
 <td>E1502</td>
 </tr>
 <tr>
 <td>5-cc syringe</td>
 <td>Becton-Dickinson</td>
 <td>309603</td>
 </tr>
 <tr>
 <td>Straight Dressing Forceps With Serrations</td>
 <td>Storz Ophthalmics</td>
 <td>E1400</td>
 </tr>
 <tr>
 <td>23 gauge needle</td>
 <td>Becton-Dickinson</td>
 <td>305145</td>
 </tr>
 <tr>
 <td>Colibri forceps</td>
 <td>Storz Ophthalmics</td>
 <td>2/132</td>
 </tr>
 <tr>
 <td>Castroviejo angled corneal scissors</td>
 <td>Storz Ophthalmics</td>
 <td>E3223</td>
 </tr>
 <tr>
 <td>Vannas Curved Capsulotomy Scissors</td>
 <td>Storz Ophthalmics</td>
 <td>E3387</td>
 </tr>
 <tr>
 <td>Weck-Cel surgical spears</td>
 <td>Medtronic</td>
 <td>0008680</td>
 </tr>
 <tr>
 <td>Westcott Curved Tenotomy Scissors</td>
 <td>Storz Ophthalmics</td>
 <td>E3320</td>
 </tr>
 </tbody>
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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.

Watch this Scientific Journal Video about Dissection of Human Vitreous Body Elements for Proteomic Analysis at JoVE.com

Dissection of Human Vitreous Body Elements for Proteomic Analysis

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 ...

dissection-of-human-vitreous-body-elements-for-proteomic-analysis

Research

JoVE Journal

Medicine

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

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.

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Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

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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.

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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 ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

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.

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Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

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Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos

Body composition is associated with risk of disease progression and treatment complications in a variety of conditions. Therefore, quantification of skeletal muscle mass and adipose tissues on Computed Tomography (CT) and/or Magnetic Resonance Imaging (MRI) may inform surgery risk evaluation and disease prognosis. This article describes two quantification methods originally described by Mourtzakis et al. and Avrutin et al.: tissue segmentation and linear measurement of skeletal muscle. Patients' cross-sectional image at the midpoint of the third lumbar vertebra was obtained for both measurements. For segmentation, the images were imported into Slice-O-Matic and colored for skeletal muscle, intramuscular adipose tissue, visceral adipose tissue, and subcutaneous adipose tissue. Then, surface areas of each tissue type were calculated using the tag surface area function. For linear measurements, the height and width of bilateral psoas and paraspinal muscles at the level of the third lumbar vertebra are measured and the calculation using these four values yield the estimated skeletal muscle mass. Segmentation analysis provides quantitative, comprehensive information about the patients' body composition, which can then be correlated with disease progression. However, the process is more time-consuming and requires specialized training. Linear measurements are an efficient and clinic-friendly tool for quick preoperative evaluation. However, linear measurements do not provide information on adipose tissue composition. Nonetheless, these methods have wide applications in a variety of diseases to predict surgical outcomes, risk of disease progression and inform treatment options for patients.

Segmentation and linear measurements quantify skeletal muscle mass and adipose tissues using Computed Tomography and/or Magnetic Resonance Imaging images. Here, we outline the use of Slice-O-Matic software and Horos image viewer for rapid and accurate analysis of body composition. These methods can provide important information for prognosis and risk stratification.

Body composition is associated with risk of disease progression and treatment complications in a variety of conditions. Therefore, quantification of ...

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.

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 ...