TITLE: Immunolabelling and clearing of intact, fixed rat spinal cord for visualization of neurons projecting to major pelvic ganglion
AUTHORS: John-Paul Fuller-Jackson, Peregrine B Osborne, Janet R Keast

[DESCRIPTION]
The whole-mount immunolabeling and clearing method (iDISCO) was used to visualize cholera toxin subunit B (CTB)-labelled lumbar and sacral preganglionic neurons in the spinal cord of the adult rat. Imaging of spinal cord was performed on a light sheet microscope with a 2x and a 12x lens. Choline acetyltransferase immunoreactivity identified preganglionic autonomic neurons and somatic motoneurons within the spinal cord.

[STEPS]
SECTION: Spinal cord preparation
1. While immersed in phosphate buffered-saline (PBS), pH 7.2, trim nerve roots of paraformaldehyde-fixed spinal cord to within approximately 2 mm of the spinal cord surface to facilitate the identification of segments later, following imaging. Segments of particular interest are T13-L2 for the lumbar preganglionic neurons, and L6-S1 for the caudal preganglionic neurons.
SECTION: Bleaching
2. Wash samples in 1x Dulbecco’s PBS (DPBS)(6 x 15 mins).
SECTION: Bleaching
3. Dehydrate samples in a series of methanol in DPBS dilutions while on rotation at 12 rpm: 
50% methanol in DPBS (1.5 h)
80% methanol in DPBS (1.5 h)
100% methanol (1.5 h)
SECTION: Bleaching
4. Bleach samples overnight in 6% hydrogen peroxide in methanol at 4°C, protected from light.
SECTION: Blocking
5. Rehydrate samples in a series of methanol in DPBS dilutions while on rotation at 12 rpm: 
100% methanol (2 x 1.5 h) 
80% methanol in DPBS (1.5 h)
50% methanol in DPBS (1.5 h)
DPBS (1.5 h)
SECTION: Blocking
6. 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
SECTION: Primary antibody incubation
7. 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.
SECTION: Secondary antibody incubation
8. Wash spinal cords in 1x DPBS with 0.5% Triton X-100 (DPBST) (6 x 15 mins).
SECTION: Secondary antibody incubation
9. 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.
SECTION: Dehydration and delipidation
10. Wash spinal cords in DPBST (6 x 15 mins).
SECTION: Dehydration and delipidation
11. Dehydrate spinal cords in a series of methanol in DPBS dilutions while on rotation at 12 rpm: 
20% methanol in DPBS (1 h)
40% methanol in DPBS (1 h)
60% methanol in DPBS (1 h)
80% methanol in DPBS (1h)
100% methanol (2 x 1 h)
SECTION: Dehydration and delipidation
12. 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.
SECTION: Dehydration and delipidation
13. Incubate samples in 100% dichloromethane for 30 mins while on rotation at 12 rpm. Repeat this step until samples sink.
SECTION: Clearing
14. 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.
SECTION: Storage
15. Store cleared samples in fresh dibenzyl ether. Keep away from light (wrap in foil and store in an opaque container).
SECTION: Light sheet microscopy
16. Prior to visualization on a light sheet microscope, samples should be transferred into ethyl cinnamate, at least 3 h prior.
SECTION: Light sheet microscopy
17. 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 or T13 (depending on sample chosen) being the point of adherence to the mounting platform.
SECTION: Light sheet microscopy
18. In order to visualize the axons and dendrites of preganglionic neurons, use the highest magnification lens available on the light sheet microscope. 
For example, a 12x lens is adequate for dendrite visualization. 
Look for cholera toxin subunit B labelled projections extending from the intermediolateral nuclei to the dorsal commissural nucleus, as well as projections into the ventral horn of the grey matter. 
SECTION: Light sheet microscopy
19. Image acquisition was always performed with 2 µm z-step between each image. Where necessary, mosaic image acquisition was applied to capture the entire region of interest, with 10% overlap between image stacks.