Revision as of 20:06, 2 December 2013

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 and sample preparation (Day 1)

• Make or thaw the hydrogel monomer solution
• Complete transcardial perfusion with the hydrogel solution (if possible)
• Incubate sample in hydrogel solution at 4°C to let the hydrogel monomers diffuse inside the tissue

Step 2: Hydrogel solution incubation (Day 1-3)

• Continue sample incubation at 4°C
  • Note: Incubation can be continued an extra day if desired. 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 (Day 3)

• De-gas the sample container
• Incubate the sample at 37°C for about 3 hours
• Remove excess gel from the sample surface (unless leaving out bis-acrylamide from the hydrogel solution)
  • Note: If sectioning the sample, it is best to complete this immediately after hydrogel embedding before placing the sample in clearing solution.
• Place sample in 50 mL of clearing solution to wash out excess unreacted monomers from the tissue (37°C or room temperature, shaking preferable)

Step 4: Clearing solution wash (Day 4)

• Replace 50 mL clearing solution and continue incubation at 37°C or room temperature
  • Note: Samples can be stored indefinitely in the clearing solution. The tissue will clear slowly from passive diffusion of SDS.

Step 5: Electrophoretic tissue clearing (ETC) (Days 5-14)

• Set up electrophoretic tissue clearing (ETC)
• 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 9 days (or more). The number of days listed represents one possibility and is primarily meant to indicate that ETC will run for a span of several days.

Step 6: PBST buffer wash (Days 14-16)

• Remove sample from the ETC chamber when it appears see-through (sample will be swollen to a larger size and not visually transparent yet)
• Place sample in 50 mL PBST (0.1% TritonX in 1X PBS) at room temperature on a shaker plate overnight to wash out SDS micelles
• Replace PBST buffer and continue incubation at room temperature with shaking overnight
  • Note: Samples can be stored indefinitely in PBST buffer. Store samples at room temperature or 4°C.

===Step 7: Immunostaining (if necessary) (Days 16-21)

• • Note: