CLARITY Timeline


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CLARITY is a multi-step process that occurs over several days. Throughout the process, the sample is washed in several different solutions that each serve a unique purpose for embedding, clearing, staining, and imaging the tissue. The following timeline outlines the order of the individual steps and presents the general day-by-day procedure. As noted, some steps may require extra days depending on the type of tissue sample and the ETC set-up. In general, while the order of the steps is important, the timeline has room for adjustment.


Step 1: Hydrogel solution and sample preparation (<1 day)

Step 2: Hydrogel solution incubation (2-3 days)

  • Incubate sample at 4°C to let the hydrogel monomers diffuse inside the tissue, shaking optional
    • Note: Longer incubation times (1-2 weeks) will probably be needed for non-perfused tissue. Do not store samples indefinitely in the hydrogel solution as polymerization will slowly occur over time.
  • Prepare clearing solution

Step 3: Hydrogel tissue embedding (<1 day)

Step 4: Clearing solution wash (2 days minimum)

  • Incubate sample in clearing solution overnight to wash out excess unreacted monomers from the tissue (37°C or room temperature, shaking preferable)
  • Replace 50 mL clearing solution and continue incubation at 37°C or room temperature
    • Note: Washes from the first 2 days contain toxic hydrogel monomers and must be disposed of as waste. Afterwards, clearing solution is safe for sink disposal.
  • Repeat clearing solution washing as desired or store in clearing solution. The tissue will clear slowly from passive diffusion of SDS micelles.
    • Note: The duration of passive clearing is dependent on factors such as the size/thickness of the tissue sample and the incubation temperature. Clearing occurs faster for smaller, thinner samples and at higher incubation temperatures (37-50°C). Gentle shaking and routinely replacing the clearing solution may also help quicken the passive clearing process.
    • Note: Samples can be stored indefinitely in the clearing solution.

Step 5: Electrophoretic tissue clearing (ETC) (~2-6 days)

  • Set up electrophoretic tissue clearing (ETC) supplies
  • Add sample to the ETC chamber and turn on clearing solution circulation and power supply
  • Run ETC to clear the lipids from the embedded tissue
  • Routinely check the clearing solution pH and replace with fresh solution if it starts to dip below ~7.5
  • Visually check the sample for clearing
    • Note: The duration of ETC depends greatly on the sample conditions (size, tissue type, etc) and the ETC conditions (temperature, voltage). This could take as little as 2-3 days or as long as 6 days (or more).
    • Note: A shorter duration of ETC clearing (1-3 days) combined with passive clearing before and/or after ETC may provide better results than ETC clearing alone.

Step 6: PBST buffer wash (2 days minimum)

  • Remove sample from the ETC chamber or clearing solution (passive clearing) when it appears see-through (sample will be swollen to a larger size and not entirely visually transparent)
  • Place sample in 50 mL PBST (0.1% TritonX in 1X PBS) at room temperature or 37°C on a shaker plate overnight to wash out SDS micelles
  • Replace PBST buffer and continue incubation with shaking overnight
    • Note: Samples can be stored indefinitely in PBST buffer. Store samples at room temperature or 4°C. The samples will remain swollen and possibly turn cloudier than they appeared in clearing solution.
    • Note: TritonX is important for washing out the SDS micelles and preventing precipitation later in the mounting solution. Do not substitute PBS for PBST when transferring the sample from clearing solution.

Step 7: Immunostaining (6+ days)

  • Incubate sample in primary antibody solution at 37°C with shaking (~2 days for 1 mm thick slice)
  • Wash sample with buffer (~1 day for 1 mm thick slice)
  • Incubate sample in secondary antibody solution at 37°C with shaking (~2 days for 1 mm thick slice)
  • Wash sample with buffer (~1 day for 1 mm thick slice)
    • Note: The immunostaining procedure and results depend on the specific antibody, so condition optimization or alternative antibodies may be required. Greater antibody concentrations and longer incubation times are needed for effective immunostaining. Starting with a 1:50 primary and secondary antibody dilution in PBST is recommended and can be adjusted as needed. The given incubation times are good starting points for 1 mm thick tissue samples, but will need to be extended for thicker samples. Tissue sections less than 2 mm thick can be stained reliably, but anything thicker than this may not get full antibody penetration into the core of the sample even with extended incubation times (6 weeks for a whole mouse brain).

Step 8: Sample imaging (0-3 days)

  • Incubate sample in mounting solution for several hours (or overnight) at room temperature or 37°C with shaking
    • Note: While 85% glycerol or other solutions with a refractive index of ~1.45 seem to optically match the refractive index of the embedded tissue, the sample still exhibits a bit of cloudiness in these solutions, especially with thicker samples such as a whole mouse brain. For the most visually transparent tissue that will allow deep penetration with the microscope, FocusClear is the best choice.
  • When the sample appears visually transparent and back to its anatomical size, mount the sample in a sealed chamber with FocusClear
  • Complete sample imaging
  • Remove the sample from the sealed imaging chamber immediately after imaging and place in PBST buffer for storage or to prepare for a second round of staining
    • Note: Sample storage in FocusClear is not recommended. An irreversible white precipitate may develop inside the hydrogel after several days.

Step 9: Multiple staining rounds (if desired)

  • After removing sample from imaging chamber, place in 50 mL PBST at room temperature or 37°C overnight with shaking to wash out imaging solution
  • Place sample in 50 mL clearing solution and incubate at 60°C overnight with shaking to wash out the first round of antibodies
  • Place sample in 50 mL PBST and incubate at room temperature or 37°C with shaking for 1-2 days to wash out the clearing solution
  • Repeat Steps 7 and 8 with the next round of antibodies