Table 1.

Free of acrylamide sodium dodecyl sulphate (SDS)‐based tissue‐clearing (FASTClear) protocol

Step	Time
1. Fixation in 10% neutral‐buffered formalin/4% paraformaldehyde (PFA)	Minimum of 3 days at 4°C
• N.B. fixation time depends on size of fresh tissue block. Typically, a 1‐cm‐thick block will take around 3 days to be fully fixed.
• This step is only required for fresh tissue. Proceed to Step 2 for formalin‐fixed tissue.
Caution: use a tightly sealed container (±parafilm) for fixation as formalin/PFA is toxic.
2. Dissect into smaller block
• A maximum of 3 mm in thickness is recommended due to immunolabelling diffusion and confocal objectives working distance limits. Note that the sectioning surface should be as flat as possible and designed to be the future imaging surface.
3. Immerse in 4% SDS buffer	Minimum of 5 days at 50°C oven
• This step improves antibody labelling and it is recommended the tissue is immersed in SDS buffer until transparency is reached. For prolonged fixed tissue (>2 years in fixation), a 2‐mm‐thick block can reach transparency in 3 months.•[However, do note that complete transparency of tissue is not a necessity as tissue will become transparent at the final refractive index matching step]
• Frequent change in buffer (daily to twice weekly) can improve the speed of reaching transparency.
4. Washing in 0.1% phosphate‐buffered saline (PBS)‐Triton	3 × 1 h at 50°C
5. Blocking and permeabilization in blocking medium [0.6 M glycine, 0.2% Triton X‐100, 6% Donkey serum, 20% dimethyl sulfoxide (DMSO) in PBS]	Overnight at 37°C
• Add enough blocking medium to cover the tissue.
• Optional if the antibody is known to be of high specificity.
6. Washing in 0.1% PBS‐Triton	2 x 1 h at 37°C
7. Primary antibody incubation (diluted in 0.2% Tween‐20, 5% DMSO, 3% Donkey serum, 0.01% sodium azide in PBS)	Minimum of 2 days at 37°C
• Start with a low concentration (e.g. 1:1000; 2 μl in 2 ml of diluent), supplementing antibody daily/twice daily until a final concentration of around 1:50–1:100 is reached.
• Optimal concentration and days of incubation vary between antibodies.
• As an example, tyrosine hydroxylase antibodies (Millipore AB152) can reach complete penetration to a depth of 2 mm on each side in 3 days at a final concentration of 1:100.
• If multiple antigen labelling is required, it is recommended to perform immunolabelling sequentially.
8. Washing in 0.1% PBS‐Triton	3 × 1 h at 37°C; then overnight at 37°C
9. Secondary antibody incubation (diluted in 0.2% Tween‐20, 5% DMSO, 3% Donkey serum, 0.01% sodium azide in PBS)	Minimum of 2 days at 37°C
• Same as Step 7 above.
• 4′,6‐Diamidino‐2‐phenylindole or fluorophore‐conjugated lectin can be added at this stage (1:100 from a stock of 1 μg/ml diluted with 1:1 water: DMSO) for better tissue orientation.
10. Washing in 0.1% PBS‐Triton	5 × 1 h at 37°C; then overnight at 37°C
11. Immersion in refractive index matching medium	RT until transparency is reached
• If tissue is transparent/almost transparent after Step 3
Immerse in 47% 2,2′‐thiodiethanol diluted in 0.01 M PBS without saline or 70% w/v Sorbitol in 0.1 M phosphate buffer (as previously described in ref. 3).
• If tissue is opaque after Step 3, follow 3DISCO clearing method:
Dehydrate tissue in 50% tetrahydrofuran (THF) (overnight), 70% THF (1 h), 80% THF (1 h), 100% THF (1 h) 100% THF (1 h) then dibenzyl ether until transparency is reached.
Caution: perform this step in the fume hood.