Ce3D+ and Ce3D++ Tissue Clearing

Ce3D+ and Ce3D++ Clearing Protocol 

The original Ce3D solution recipe yields a refractive index of 1.49-1.5. This protocol describes two modified Ce3D clearing solutions that adjust the refractive index of the final solution to better match the refractive index of microscopy oils (nD = 1.515). Additionally, the toxic component 1-thioglycerol is omitted. Though 1-thioglycerol slightly improves clearing, the clearing has been satisfactory without including it.

Reagents

• N-Methylacetamide ≥99%: Sigma Aldrich M26305 
• Iohexol (sold as Nycodenz, Histodenz): Accurate Chemical AN1002424
• Triton X-100: Sigma Aldrich T8787
• 1x PBS: various suppliers

If clearing and imaging large tissues/embryos:

• Low melting point agarose: Thermofisher Scientific 16520100

or

• Ultra-low gelling temperature agarose: Sigma Aldrich A5030

Stock Solution Preparations:

Preparation of N-Methylacetamide 

1. Melt the N-Methylacetamide in the original 100 mL bottle in hot water (melting point of N-Methylacetamide is 28° Celsius).
2. Aliquot 20 mL of melted N-Methylacetamide into 50 mL screw cap tubes.
3. Let the N-Methylacetamide solidify and store at room temperature for later use with the lid tightly closed. 
4. To make 40% v/v N-Methylacetamide, melt the pre-aliquotted (20 mL) N-Methylacetamide in hot water.
5. Fill tube to the 50 mL line with 1X PBS and store at room temperature.

General Protocols:

• Final solutions should be stored at room temperature.
• Keep cleared tissues in a sealed container (e.g., parafilm wrapped glass bottomed dish) to prevent evaporation. This is important because the refractive index will be affected by change in water content.
• The refractive index of the final preparation can be checked using a refractometer for quality control. We use a Reichert Technologies AR200 Automatic Digital Refractometer (#13950000).

Ce3D+ protocol

Ce3D+ is designed to be used for low-volume samples such as cultured cells. 

1. In a 50 mL screw cap tube, weigh 20 g of Nycodenz AG powder. The powder should fill the tube close to the 20 mL line.
2. In the same 50 mL tube, carefully pipette 10.5 g of 40% v/v N-Methylacetamide solution.
3. With the tube still on the scale, carefully pipette 22.5 mg (~22 µL) of Triton X-100 to yield a final concentration of 0.1% v/v.
   Note: Slightly more is not a problem, so anywhere between 22-24 mg is acceptable.
4. Mix solution overnight on a slow rotator (e.g., 4 rpm) at room temperature.
5. Aspirate all liquid from plate/slide containing the cells, apply Ce3D+, and equilibrate at room temperature.

Ce3D++ protocol 

Ce3D++ is a variant of Ce3D+ with a higher concentration of iohexol than Ce3D+, which is designed to compensate for the water initially contained in larger samples (e.g., tissues, embryos, etc.). 

For small samples (not embedded in agarose) that are 5-50 mg, which we approximate to contain 5-50 µL of water, it is a single-step process. 

1. In a 50 mL screw cap tube, weigh 20.83 g of Nycodenz AG powder. The powder should fill the tube close to the 20 mL line.
2. In the same 50 mL tube, carefully pipette 10.5 g of 40% v/v N-Methylacetamide solution.
3. With the tube still on the scale, carefully pipette 22.5 mg (~22 µL) of Triton X-100 to yield a final concentration of 0.1% v/v.
   Note: Slightly more is not a problem, so anywhere between 22-24 mg is acceptable.
4. Mix the solution overnight on a slow rotator (e.g., 4 rpm) at room temperature.
5. Incubate the sample at room temperature overnight in the volume of Ce3D++ solution as calculated below:
   1. Volume of Ce3D++ = Mass of sample (g) * 24.1 (mL/g) = ____ mL
      1. For example, if your sample is weighs 0.007 g, incubate in ~169 µL of Ce3D++ solution overnight.
         Note: Gentle rocking can be used to speed up equilibration. 
6. The sample must be imaged in the solution that it was incubated in, and not fresh solution.

For larger samples (e.g., embedded in 1.5% low melt or ultralow melt agarose), it is a two-step process where solution must be exchanged once. 

1. In a 50 mL screw cap tube, weigh 20.83 g of Nycodenz AG powder. The powder should fill the tube close to the 20 mL line.
2. In the same 50 mL tube, carefully pipette 10.5 g of 40% v/v N-Methylacetamide solution.
3. With the tube still on the scale, carefully pipette 22.5 mg (~22 µL) of Triton X-100 to yield a final concentration of 0.1% v/v.
   Note: Slightly more is not a problem, so anywhere between 22-24 mg is acceptable.
4. Mix the solution overnight on a slow rotator (e.g., 4 rpm) at room temperature.
5. Incubate sample (including the agarose that the sample is embedded in) in the following volume of Ce3D++ solution twice (aspirated and replaced once by the same volume) at room temperature. Make sure that one incubation is at least overnight; the other incubation can be a few hours.
   1. If volume of the sample (+ agarose) is known:
      1. Volume of Ce3D++ = Volume agarose (mL) * 4 = ____ mL
      2. For example, if your sample is embedded in 400 µL agarose, incubate first in 1.6 mL of Ce3D++ solution, then aspirate the 1.6 mL (to leave only the sample in agarose), and incubate again in a fresh 1.6 mL of Ce3D++ solution. 
      3. If the volume of the sample (+ agarose) is unknown, weigh the sample and estimate the volume assuming the density of water.
         Note: Gentle rocking can be used to speed up equilibration. 
6. The sample must be imaged in the solution that it was last incubated in, and not fresh solution.