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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially 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) – bilateral star-shaped structures of the sympathetic chain – are an important component of the sympathetic infrastructure. The SG are a recognized target for 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.

Wprowadzenie

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.

Protokół

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'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 /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.

  1. Prepare 50 mL of ice-cold (3-4 °C) heparinized (20 units/mL, see Table of Materials) phosphate-buffered saline (PBS). Perform dissection of the SG at room temperature (RT).
  2. 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 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.
  3. 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 ribcage.
  4. 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.
  5. Flush the thorax thoroughly with heparinized PBS using a plastic disposable pipette until all traces of blood are removed.
  6. Place the torso under stereomicroscope preparation binoculars and ensure good lighting in the thorax with external light sources.
  7. 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.
  8. Gently use the tip of the Dumont #5/45 forceps to expose the connective tissue lateral to the longus colli muscle.
  9. Turn the forceps around by 180° and use the flat side to grip the SG and pull it out with minimal pressure.
  10. Repeat with the second SG.
  11. 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.
  12. Depending on the goal of the experiment, proceed with section 2, 3, or 5 of this protocol.

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 μL (for antibody-containing solutions) to 200 μL (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 μL pipette with an additional 10 μL tip on top of the 200 μL tip. This will prevent aspiration of the SG in the pipette tip. Use freshly prepared solutions and sterile liquids to prevent bacterial growth.

  1. Fix SG for histology for 2 h at RT in 4% methanol-free paraformaldehyde (PFA)/PBS.
  2. Process SG as quickly as possible, but they can be stored for 2-4 weeks at 4-6 °C in PBS with 0.02% (w/v) sodium azide at this point.
  3. 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.
  4. 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.
  5. Treat SG with Dent's bleach to improve antibody permeabilization31. Freshly prepare Dent'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 μL per SG and place the plate on an orbital shaker for 1 h at RT.
  6. 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.
  7. Perform permeabilization by incubating SG twice for 60 min each in PBS/1% Triton-X-100 at RT.
  8. Remove permeabilization solution from the SG and add Sudan black working solution. Incubate for 2 h at RT on an orbital shaker.
  9. 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.
  10. 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.
  11. Add 200 μL of PBS/0.1% Triton-X-100 (PBS-T) and wash for 5 min at RT on an orbital shaker.
  12. Remove PBS-T by aspirating it with a pipette and repeat 2 times.
  13. Remove PBS; add 200 μL of blocking solution and incubate at 4 °C overnight on an orbital shaker.
  14. 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).
  15. Perform primary antibody incubation for 36-48 h at 4 °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.
  16. Remove antibody solution carefully and add 200 μL of PBS-T. Place the plate on an orbital shaker for 30 min.
  17. Remove PBS-T and repeat the washing step 5 additional times.
  18. 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 μg/mL of bisbenzimide H33342 trihydrochloride (Hoechst staining) if nuclear staining is desired. Incubate for 12-24 h at 4 °C on an orbital shaker.
  19. Remove the antibody solution carefully and add 200 μL of PBS-T. Place the plate on an orbital shaker for 30 min.
  20. Remove PBS-T and repeat the washing step 5 additional times. For the last step, use PBS without Triton.
  21. For embedding, spread 50-100 μL 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.
  22. Use Dumont #5/45 forceps to correct positioning with the appropriate magnification.
  23. 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.
  24. 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 °C.

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 μL of buffers and solutions.

  1. Perform dissection of the SG as described in steps 1.1-1.13.
  2. Fixate SG for 1 h in 200 μL of 4% MeOH-free PFA/PBS in one well of a 96-well plate placed on an orbital shaker.
  3. Wash SG three times for 30 min each in 0.1% Tween-20/PBS on an orbital shaker.
  4. 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.
  5. Store SG at -20 °C in 100% MeOH overnight.
  6. The next day, pre-warm the incubator to 40 °C. Check the temperature with a thermometer.
  7. Rehydrate SG in reverse MeOH/PBS series (100% MeOH, 70% MeOH/PBS, 50% MeOH/PBS) for 10 min each.
  8. Wash SG three times for 5 min each in PBS.
  9. In the meantime, start pre-warming the probes by incubation at 40 °C for 10 min, followed by cooling for 10 min.
  10. Incubate SG in 200 μL of Protease III for 15 min.
  11. 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.
  12. Wash SG in 200 μL of 0.1% Tween-20/PBS 3x for 5 min each on an orbital shaker.
  13. Optional: If co-staining with several probes is desired, dilute Channel-2 (50x) and Channel-3 probe (50x) in Channel-1 probe (1x).
  14. Cover SG with 100 μL of probe for the gene of interest and incubate overnight at 40 °C with slight agitation. Place the 96-well plate in a wet chamber at 40 °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.
  15. Wash SG in supplied washing buffer 3x for 15 min each on an orbital shaker.
  16. 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.
  17. Re-fix SG for 10 min at RT in 4% PFA/PBS on an orbital shaker.
  18. Wash SG in 200 μL of supplied washing buffer 3x for 5 min each on an orbital shaker at RT.
  19. For amplification, incubate SG with 100 μL of Amp1 for 35 min at 40 °C on an orbital shaker.
  20. Carefully remove any liquid and wash SG in 200 μL of supplied washing buffer 3x for 5 min each at RT on an orbital shaker.
  21. Incubate SG with 100 μL of Amp2 for 35 min at 40 °C on an orbital shaker.
  22. Repeat step 3.18.
  23. Incubate SG with 100 μL of Amp3 for 20 min at 40 °C on an orbital shaker.
  24. Repeat step 3.18.
  25. Incubate SG with 100 μL of supplied Multiplex FL v2 HRP-C1 for 20 min at 40 °C on an orbital shaker.
  26. Repeat step 3.18.
  27. Prepare Opal-conjugated secondary antibody 1:1,000 in 200 μL of supplied TSA-buffer and incubate SG for 35 min at 40 °C on an orbital shaker. Protect from light during incubation and from this step on.
  28. Repeat step 3.18.
  29. Incubate SG in 100 μL of supplied Multiplex FL v2 HRP-blocker for 15 min at 40 °C on an orbital shaker.
  30. Repeat step 3.18.
  31. Optional: For co-staining with Channel-2 probe, repeat steps 3.25-3.30 with supplied Multiplex FL v2 HRP-C2.
  32. Optional: For co-staining with Channel-3 probe, repeat steps 3.25-3.30 with supplied Multiplex FL v2 HRP-C3.
  33. In case of subsequent immunofluorescent staining, perform steps 2.13-2.25 of this protocol.
  34. Incubate in 1% BSA/PBS for 30 min on an orbital shaker.
  35. Incubate SG for 30 min in 1 μg/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.
  36. Repeat step 3.18.
  37. Embed as described in steps 2.19-2.22.

4. Imaging and analyses of murine stellate ganglia

  1. Perform confocal microscopy of embedded SG in the local imaging facility.
  2. 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.
  3. 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.
  4. Using this method, perform measurement of approximately 100 cells per SG.
  5. 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.

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).

  1. Prepare a 2 mL tube with 500 μL 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.
  2. Perform dissection of the SG as described in steps 1.1-1.13.
  3. Immediately immerse both SG directly in one tube with phenol/guanidine thiocyanate solution and shock-frost tube in liquid nitrogen.
  4. Store at -80 °C until further processing.
  5. 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 4 °C.
  6. Centrifuge tubes at 500 x g for 1 min at 4 °C so that SG are at the bottom of the tube.
  7. Put tubes into the metal parts of the Tissue Lyser and lyse for 1 min at 20 Hz.
  8. Repeat steps 5.6 and 5.7 up to 5 times until no intact tissue is detectable.
  9. Transfer the liquid into a fresh 1.5 mL tube.
  10. Perform RNA isolation with a column-based RNA isolation kit (e.g., miRNeasy mini kit) according to the manufacturer's instructions.
  11. Elute RNA in 20 μL 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 μL 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.
  12. 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's instructions.
  13. Dilute to a final concentration of 2.5 ng/μL 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.
  14. 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.

Wyniki

Figure 1 visualizes how to identify and dissect the SG. Figure 1A shows a schematic drawing of the location, while Figure 1B presents the view into 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 ...

Dyskusje

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 – a murine element within this network – and perform subsequent analyses on RNA, protein, and cellular level.

Ujawnienia

The authors have nothing to disclose.

Podziękowania

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].

Materiały

NameCompanyCatalog NumberComments
96-well plateTPP92097RNAscope
Adhesion Slides SuperFrost plus  25 x 75 x 1 mmR. Langenbrinck03-0060Microscopy
Albumin bovine Fraction V receptor grade lyophil.Serva11924.03Whole mount staining
bisBenzimide H33342 trihydrochloride (Hoechst)Sigma-Aldrich, St. Louis, MO, USAB2261Whole mount staining
Chicken anti neurofilamentEMD MilliporeAB5539Whole mount staining
Dimethyl sulfoxide (DMSO)Merck, KGA, Darmstadt, GermanyD8418Whole mount staining
Donkey anti chicken IgY Alexa 647 Merck, KGA, Darmstadt, GermanyAP194SA6Whole mount staining
Donkey anti goat IgG Alexa 568 Thermo Fisher ScientificA11057Whole mount staining
Donkey anti rabbit IgG Alexa 488 Thermo Fisher ScientificA21206Whole mount staining
Drying block 37-100 mmWhatman (Sigma Aldrich)WHA10310992 Whole mount staining
Eosin YSigma AldrichE4009Whole mount staining
Ethanol 99 % denatured with MEK, IPA and Bitrex (min. 99,8 %)Th.Geyer2212.5000Whole mount staining
Eukitt mounting mediumAppliChem253681.0008Whole mount staining
Fluoromount-GSouthern Biotech0100-01Whole mount staining
Fluoromount-G + DAPISouthern Biotech0100-20Whole mount staining
Goat anti choline acetyltransferaseEMD MilliporeAP144PWhole mount staining
H2O2 30% (w/w)Merck, KGA, Darmstadt, GermanyH1009Whole mount staining
Heparin Sodium 25.000 UI / 5mlRotexmedicaPZN: 3862340Preparation SG
High-capacity cDNA reverse transctiption kitLife technologies 4368813RNA isolation
Isoflurane (Forene)Abbott Laboratories2594.00.00Preparation SG
Mayer's hemalum solutionMerck1.09249.0500Whole mount staining
MethanolSigma-Aldrich34860Whole mount staining
Microscope cover glasses 20x20 mm or smallerMarienfeld0101040Whole mount staining
miRNeasy Mini KitQiagen217004RNA isolation
NanoDrop 2000cThermo Fisher ScientificND-2000CRNA isolation
Opal 570 Reagent PackAkoya BioscienceFP1488001KTRNAscope
Paraformaldehyde, 16% w/v aq. soln., methanol free Alfa Aesar43368Whole mount staining
Pasteur pipettes, LDPE, unsterile, 3 ml, 154 mmTh.Geyer7691202Whole mount staining
Phosphate-buffered saline tabletsGibco18912-014Whole mount staining
Pinzette Dumont SS ForcepsFineScienceTools11203-25Preparation SG
QIAzol Lysis ReagentQiagen 79306RNA isolation
Rabbit anti tyrosine hydroxylaseEMD MilliporeAB152Whole mount staining
RNAlaterMerckR0901-100MLRNA isolation (optional)
RNAscope Multiplex Fluorescent Reagent Kit v2biotechne (ACD)323100RNAscope
RNAscope Probe-Mm-S100b-C2biotechne (ACD)431738-C2RNAscope
RNAscope Probe-Mm-Tubb3biotechne (ACD)423391RNAscope
Stainless steel beads 7 mm Qiagen 69990RNA isolation
Sudan black BRoth0292.2Whole mount staining
TaqMan Gene Expression Assay Cdkn1b (Mm00438168_m1)Thermo Fisher Scientific4331182Gene expression analysis
TaqMan Gene Expression Assay Choline acetyltransferase (Mm01221880_m1)Thermo Fisher Scientific4331182Gene expression analysis
TaqMan Gene Expression Assay MKi67 (Mm01278617_m1)Thermo Fisher Scientific4331182Gene expression analysis
TaqMan Gene Expression Assay PTPCR (Mm01293577_m1)Thermo Fisher Scientific4331182Gene expression analysis
TaqMan Gene Expression Assay S100b (Mm00485897_m1)Thermo Fisher Scientific4331182Gene expression analysis
TaqMan Gene Expression Assay Tyrosin Hydroxylase (Mm00447557_m1)Thermo Fisher Scientific4331182Gene expression analysis
TaqMan mastermixApplied biosystems4370074Gene Expression analysis 
Tissue Lyser IIQiagen85300RNA isolation
Triton X-100 10% solutionSigma-Aldrich93443-100mlWhole mount staining
Tween-20Sigma-AldrichP9416-100MLRNAscope
Wacom bamboo penWacomCTL-460/KCell size measurements
Whatman prepleated qualitative filter paper, Grade 595 1/2Sigma-AldrichWHA10311647Whole mount staining
Wheat Germ Agglutinin, Alexa Fluor 633 ConjugateThermo Fisher ScientificW21404RNAscope

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