Jun 18, 2026

Rabbit DRG Dissection Protocol

DOI
https://dx.doi.org/10.17504/protocols.io.261geqondg47/v1
  • Brendan Moline1,
  • Elvia Mena Avila1,
  • Emily Reedich1,
  • Landon Genry1,
  • Megan Detloff2,
  • Katharina Quinlan1
  • 1University of Rhode Island;
  • 2Drexel University
Katharina Quinlan
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DOI: https://dx.doi.org/10.17504/protocols.io.261geqondg47/v1
Protocol Citation: Brendan Moline, Elvia Mena Avila, Emily Reedich, Landon Genry, Megan Detloff, Katharina Quinlan 2026. Rabbit DRG Dissection Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.261geqondg47/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 16, 2026
Last Modified: June 18, 2026
Protocol  Integer ID: 319206
Keywords: dorsal root ganglia, patch clamp, New Zealand White rabbit, rabbit dorsal root ganglion cell, rabbit drg dissection protocol, whole cell patch clamp, live sensory afferent, different classes of live sensory afferent, protocols from mice, new zealand white rabbit
Funders Acknowledgements:
NIH NINDS
Grant ID: NS135580
Abstract
We have developed this protocol in order to perform whole cell patch clamp on rabbit dorsal root ganglion cells. This allows assessment of electrical activity in different classes of live sensory afferents. In order to achieve this, we have modified protocols from mice and human studies, to tailor them to New Zealand White rabbits aged postnatal days 5 and 18.
Guidelines
This protocol requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee. The preparation and cell washing & mounting steps are to be performed in a biosafety cabinet to maintain proper sterility of cells once isolated.
Materials
1. Poly-L-lysine hydrobromide (P0879-100MG; Millipore Sigma; Burlington, MA, USA)
2. Horse serum (16050130; Thermo Fisher; Waltham, MA, USA)
3. Dulbecco’s modified eagle medium (12430054; Fisher Scientific; Pittsburgh, PA, USA)
4. Trypsin (LS003703; Worthington Biochemical Corporation; Lakewood Township, NJ, USA)
5. Collagenase D (COLLD-RO; Roche; Basel, Switzerland)
6. Artificial cerebrospinal fluid (aCSF)
Contains, in mM: 125 sodium chloride, 2.5 potassium chloride, 25 D-glucose, 26 sodium bicarbonate, 1 magnesium sulfate heptahydrate, 1.25 sodium phosphate monobasic monohydrate, 2.4 calcium chloride dihydrate
7. Dissection solution
Contains, in mM: 12 sodium chloride, 2.5 potassium chloride, 25 D-glucose, 250 sucrose, 26 sodium bicarbonate, 3 magnesium sulfate heptahydrate, 1.25 sodium phosphate dibasic, 1 calcium chloride dihydrate
8. 4% paraformaldehyde in 1X phosphate buffered saline
9. 70% ethanol
10. Sylgard-lined dissection chamber
11. Parafilm
12. Dissection tools (decapitation scissors, spring scissors for cutting bone, fine spring scissors, rough forceps, fine forceps, tissue scissors)
Preparation
Place four 24 x 50 millimeter coverslips inside a 6-well plate and coat with poly-L-lysine hydrobromide.
Prepare cell culture solution by adding horse serum to Dulbecco’s modified eagle medium, so the former is present at a concentration of 10%.
Add supplemented DMEM to petri dishes. Seal with parafilm.
Add supplemented DMEM to 25 cm2 cell culture flasks.
Add trypsin and collagenase D enzymes to DMEM-containing flasks. Seal the flasks with parafilm.
Bubble fresh artificial cerebrospinal fluid (aCSF) and dissection solution with oxygen while chilled on ice.  Adjust pH to 7.4 after bubbling for one hour and keep bubbling on ice.
Use sodium hydroxide (NaOH) and potassium hydroxide (KOH) when adjusting the pH for dissection and aCSF solutions, respectively.
P5 Dissection
Euthanize the animal. Euthanasia is confirmed when toe pinch does not produce a reflexive response.
Perform secondary euthanasia via decapitation at the base of the skull. Feel for a divot where the spinal cord meets the skull.
Pin the carcass to a sylgard-lined dissection chamber ventral-side up. Add ice-cold dissection solution to the chamber until the carcass is fully submerged.
Incise the skin in the genital region to confirm the sex of the animal.
Perform an evisceration by incising the abdominal and chest cavities, and remove the organs.
Un-pin the carcass and re-pin it dorsal side up. Change dissection solution frequently throughout dissection to maintain visibility and freshness, and to keep the tissue cold. Proceed to step 29.
P18 Dissection
Euthanize the animal. Euthanasia is confirmed when toe pinch does not produce a reflexive response.
Mount the carcass on a metal grid ventral-side up and tie the forelimbs to the grid with twist ties.
Spray the upper abdominal and chest cavity with 70% ethanol.  Rub the alcohol down to prevent loose fur from coating dissection tools.
Begin the perfusion flow before beginning the dissection. The flow rate should be approximately 11 mL/min.
Make a small incision in the lower chest cavity, just below the sternum, and proceed as deep until the liver is exposed.
Widen the incision to the sides of the animal, taking care to avoid incising organs.
Incise rostrally, cutting through the diaphragm, and continue until only the uppermost rib(s) are still intact. This will expose the lungs and heart. Use a hemostat to hold the detached ribcage out of the way of the dissection.
Using forceps, carefully grip the pericardium at the apex of the heart and pull gently. Incise the pericardium at the gap that appears between the tissue and heart muscle.
Remove the pericardium to fully expose the heart chambers.
Make a small incision in the lower left ventricle near the apex.
Insert the perfusion needle into the incision. Ensure that aCSF is being dispensed from the line before insertion so air is not introduced into the circulatory system.
Make an incision in the right atrium to allow blood to drain.
Position the needle so the tip is in the aorta. Clamp the needle into position with a hemostat (clamp at location where needle is inserted into heart).
Perfuse the animal until the liver has blanched and solution being pumped from the atrium runs clear.
Once perfusion is complete, remove the animal from the perfusion grid and move to a pee pad.
Perform secondary euthanasia via decapitation at the base of the skull. Feel for a divot where the spinal cord meets the skull. Proceed to step 29.
DRG Extraction & Digestion
Using spring scissors with curved tips, carefully incise the vertebrae dorsally to the spinal cord, alternating cuts evenly between the left and right sides and keeping the tips angled up and away from the spinal cord. Use forceps to raise the cut segment of the vertebrae to expose the spinal cord. Proceed until the entire spinal cord (cervical to sacral) has been exposed.
Take care not to cut too wide, as this could potentially damage the dorsal root ganglia (DRGs).
With the spring scissors, carefully widen the exposed region as needed so the roots of DRGs are visible in the cervical and lumbar regions of the spinal cord.
To remove lumbar DRGs, carefully grip the DRG fiber (not the body) with fine forceps and cut the DRG from the spinal cord with fine spring scissors. Cut through the fiber to fully separate the DRG from the carcass.
For DRGs at the level of L6 & S1, transfer the DRG into a petri dish containing supplemented DMEM.
For DRGs at the level of L7, transfer the DRG into a 1.5 mL microcentrifuge tube containing 4% paraformaldehyde (PFA) in 1X phosphate buffered saline (PBS).
To expose cervical DRGs, use fine spring scissors with curved tips. Carefully insert the tips into the bony processes containing the DRGs (with the tips pointed up and away from the tissue) and cut through the bone. Repeat for cervical sections C6-8. Use forceps to lift the detached bone and trim it away with bone scissors to fully expose these DRGs.
To give an example, insert the tips of the scissors so they are in the processes containing the C6 & C7 DRGs, respectively, and resting above the DRGs. Squeezing the scissors will allow you to cut through the bone separating these two DRGs.
By repeating this with multiple adjacent DRGs, you can expose this entire group of DRGs at once.
To remove cervical DRGs, carefully grip the DRG fiber (not the body) with fine forceps and cut the DRG from the spinal cord with fine spring scissors. Cut through the fiber to fully separate the DRG from the carcass.
For DRGs at the level of C6 & C8, transfer the DRG into a petri dish containing supplemented DMEM.
For DRGs at the level of C7, transfer the DRG into a 1.5 mL microcentrifuge tube containing 4% PFA in 1X PBS.
Place all tubes containing PFA-fixed DRGs into a 4°C refrigerator.
Using fine forceps and fine spring scissors, gently trim white matter and any blood from the DRGs in DMEM, leaving only the gray matter.
Section the cleaned DRGs into 1-millimeter sections and transfer them into flasks containing enzyme-supplemented DMEM.
Shake the flasks in a 37°C water bath for 40 minutes to digest the DRGs.
Cell Washing & Mounting
Using 5 mL serological pipettes, transfer the digested cells to 15 mL falcon tubes containing warm, fresh DMEM.
Centrifuge the tubes for 6 minutes at 600 rpm.
While the tubes are being centrifuged, wash the poly-L-lysine coated coverslips twice with DMEM. Add DMEM to the coverslips after the second wash.
Carefully aspirate the DMEM without disturbing the pelleted cells, leaving 0.5-1 mL of DMEM in the tubes.
Add fresh DMEM and invert tubes before centrifuge for 6 minutes at 600 rpm.
Carefully aspirate the DMEM without disturbing the pelleted DRGs, leaving 0.5-1 mL of DMEM in the tubes.
Using 5 mL serological pipettes, triturate the pelleted cells & remaining DMEM 30 times. Then, using 1000 µL micropipettes, triturate the cell/DMEM mixture 30 times.
Remove DMEM from the coated coverslips and transfer cells to coverslips. Let cells rest for 30 minutes.
Carefully flood wells with fresh DMEM. Transfer well plate to a CO₂ incubator set to 37°C and 5.0% CO₂.
Cells will be ready to record after one hour.
Protocol references
North, R. Y., Li, Y., Ray, P., Rhines, L. D., Tatsui, C. E., Rao, G., Johansson, C. A., Zhang, H., Kim, Y. H., Zhang, B., Dussor, G., Kim, T. H., Price, T. J., & Dougherty, P. M. (2019). Electrophysiological and transcriptomic correlates of neuropathic pain in human dorsal root ganglion neurons. Brain : a journal of neurology, 142(5), 1215–1226. https://doi.org/10.1093/brain/awz063

Odem, M. A., Bavencoffe, A. G., Cassidy, R. M., Lopez, E. R., Tian, J., Dessauer, C. W., & Walters, E. T. (2018). Isolated nociceptors reveal multiple specializations for generating irregular ongoing activity associated with ongoing pain. Pain, 159(11), 2347–2362. https://doi.org/10.1097/j.pain.0000000000001341

Stucky, C. L., & Lewin, G. R. (1999). Isolectin B(4)-positive and -negative nociceptors are functionally distinct. The Journal of neuroscience : the official journal of the Society for Neuroscience, 19(15), 6497–6505. https://doi.org/10.1523/JNEUROSCI.19-15-06497.1999

Wu, Z. Z., & Pan, H. L. (2004). High voltage-activated Ca(2+) channel currents in isolectin B(4)-positive and -negative small dorsal root ganglion neurons of rats. Neuroscience letters, 368(1), 96–101. https://doi.org/10.1016/j.neulet.2004.06.067