Jan 07, 2026

Immunolabelling and clearing of intact, fixed rat spinal cord for visualization of motor nuclei

 Forked from Immunolabelling and clearing of intact spinal cord for visualization of lower urinary tract afferents
DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwjq47lmk/v1
Immunolabelling and clearing of intact, fixed rat spinal cord for visualization of motor nuclei
  • 1University of Melbourne
Janet R Keast
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwjq47lmk/v1
Protocol Citation: John-Paul Fuller-Jackson, Peregrine B Osborne, Janet R Keast 2026. Immunolabelling and clearing of intact, fixed rat spinal cord for visualization of motor nuclei. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjq47lmk/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: March 05, 2023
Last Modified: January 07, 2026
Protocol  Integer ID: 78162
Keywords: neural tracing, retrograde tracing, clearing, idisco, light sheet microscopy, rat spinal cord for visualization, motoneuron nuclei in the lumbosacral spinal cord, imaging of spinal cord, lumbosacral spinal cord, spinal cord, motoneuron nuclei, somatic motoneuron, immunolabelling, neuron, majority of neuron, autonomic preganglionic neuron, motor nuclei, imaging, cytoarchitectural context
Funders Acknowledgements:
NIH SPARC
Grant ID: 3OT2OD023872
Abstract
The whole-mount immunolabeling and clearing method (iDISCO) was used to visualize motoneuron nuclei in the lumbosacral spinal cord of adult rats. Imaging of spinal cord was performed on a light sheet microscope with a 12x lens. Choline acetyltransferase immunolabelling identified somatic motoneurons and autonomic preganglionic neurons, while NeuN immunolabelling identified a majority of neurons, to provide a cytoarchitectural context.
Materials
Materials
MethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641
DichloromethaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #320269
Dibenzyl etherMerck MilliporeSigma (Sigma-Aldrich)Catalog #108014
Ethyl cinnamateMerck MilliporeSigma (Sigma-Aldrich)Catalog #112372
1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
Gelatin from porcine skinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1890
Hydrogen peroxide 30%Merck Millipore (EMD Millipore)Catalog #822287.1000
SaponinMerck MilliporeSigma (Sigma-Aldrich)Catalog #S4521
ThimerosalMerck MilliporeSigma (Sigma-Aldrich)Catalog #T5125
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
Anti-NeuN Antibody, clone A60Merck Millipore (EMD Millipore)Catalog #MAB377
Goat anti-choline acetyltransferase antibodyMerck Millipore (EMD Millipore)Catalog #AB144P
Cy3 donkey anti-mouse IgGJackson ImmunoResearch Laboratories, Inc.Catalog #715-165-150
AF647 donkey anti-goat IgGJackson ImmunoResearch Laboratories, Inc.Catalog #705-605-147
Equipment
Equipment
Ultramicroscope II
NAME
Light sheet microscope
TYPE
Miltenyi Biotec
BRAND
NA
SKU
https://www.miltenyibiotec.com/AU-en/products/ultramicroscope-ii.html
LINK

Solutions
PBS: phosphate-buffered saline, 0.1 M, pH 7.2
DPBS: 1x Dulbecco's phosphate-buffered saline
DPBS-T: 1x Dulbecco's phosphate-buffered saline containing 0.5% Triton X100
DPBSG-T: 1x Dulbecco's phosphate-buffered saline containing 0.2% gelatin, 0.5% Triton X-100 and 0.01% thimerosal
Primary antibodies

ABCDE
AbbreviationSynonymRRIDHost speciesDilution
ChATCholine acetyltransferaseAB_11214092Goat1:500
NeuNFox3AB_2298772Mouse1:2000

Secondary antibodies

ABCD
Tag-antibodyHost speciesRRIDDilution
AF647 anti-goatDonkeyAB_23404371:2000
Cy3 anti-mouseDonkeyAB_23408131:1000

Spinal cord preparation
30m
While immersed in phosphate buffered-saline (PBS), pH 7.2, trim nerve roots of fixed spinal cord to within approximately 2 mm of the spinal cord surface to facilitate the identification of segments later, following imaging.
Bleaching
1d
Wash samples in 1x Dulbecco’s PBS (DPBS)(6 x 15 mins).
Dehydrate samples in a series of methanol in DPBS dilutions while on rotation at 12 rpm:
  1. 50% methanol in DPBS (1.5 h)
  2. 80% methanol in DPBS (1.5 h)
  3. 100% methanol (1.5 h)
Bleach samples overnight in 6% hydrogen peroxide in methanol at 4°C, protected from light.
Blocking
2d
Rehydrate samples in a series of methanol in DPBS dilutions while on rotation at 12 rpm:
  1. 100% methanol (2 x 1.5 h)
  2. 80% methanol in DPBS (1.5 h)
  3. 50% methanol in DPBS (1.5 h)
  4. DPBS (1.5 h)
Incubate samples in DPBS containing 0.2% gelatin, 0.5% Triton X-100 and 0.01% thimerosal (DPBSG-T) for 36 h while on rotation at 12 rpm
Primary antibody incubation
1w 3d
Incubate samples in primary antibody solution containing DPBSG-T with 0.1% saponin for 10 days at 37°C with agitation. Volume of solution need only be sufficient to cover the sample.
Secondary antibody incubation
5d
Wash spinal cords in 1x DPBS with 0.5% Triton X-100 (DPBST) (6 x 15 mins).
Incubate samples in secondary antibody solution containing DPBSG-T with 0.1% Saponin for 4 days at 37°C with agitation. Volume of solution need only be sufficient to cover the sample.
Dehydration and delipidation
1d 8h
Wash spinal cords in DPBST (6 x 15 mins).
Dehydrate spinal cords in a series of methanol in DPBS dilutions while on rotation at 12 rpm:
  1. 20% methanol in DPBS (1 h)
  2. 40% methanol in DPBS (1 h)
  3. 60% methanol in DPBS (1 h)
  4. 80% methanol in DPBS (1h)
  5. 100% methanol (2 x 1 h)
Incubate samples in a solution of 2/3 dichloromethane and 1/3 methanol overnight on rotation. Ensure that samples sink to the bottom of the vial at the end of this step, otherwise continue incubation in freshly made solution.
Incubate samples in 100% dichloromethane for 30 mins while on rotation at 12 rpm. Repeat this step until samples sink.
Clearing
2h
Incubate samples in dibenzyl ether until the samples have become clear. Ensure each vial is completely filled with dibenzyl ether to minimize sample oxidation as a result of large amounts of air in the vial. This process should not take longer than 2 h.
Storage
Store cleared samples in fresh dibenzyl ether. Keep away from light (wrap in foil and store in an opaque container).
Light sheet microscopy
Prior to visualization on a light sheet microscope, samples should be transferred into ethyl cinnamate, at least 3 h prior.
Remove sample from ethyl cinnamate and gently dry on a tissue. Affix sample to plastic mount using the minimum amount of super glue required. Tips:
  • Avoid adhering the sample to the base via any region of the sample that is of interest; both the super glue and proximity to the plastic will reduce imaging quality in that area.
  • Mount the sample perfectly in the middle; the light sheet microscope stage has limited mobility, particularly at higher magnifications.
  • Orientate the sample such that the thinnest plane of the sample is perpendicular to the light sheet beams. The further light has to travel through a sample, the poorer the image quality.
  • Orientate the sample so that the region of interest is facing as close to the lens as possible.
In this protocol, the spinal cord was mounted perpendicular to the light sheet beams, with the dorsal horn facing the lens, and the ventral side of L5 being the point of adherence to the mounting platform.
Protocol references
Renier, N., Wu, Z., Simon, D.J., Yang, J., Ariel, P., Tessier-Lavigne, M., 2014. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910. https://doi.org/10.1016/j.cell.2014.10.010 Belle, M., Godefroy, D., Couly, G., Malone, S.A., Collier, F., Giacobini, P., Chédotal, A., 2017. Tridimensional Visualization and Analysis of Early Human Development. Cell 169, 161-173.e12. https://doi.org/10.1016/j.cell.2017.03.008