CLARITY Timeline
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Revision as of 00:50, 19 February 2014 by Kristin Engberg (Talk | contribs)
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 (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)
- Incubate sample in primary antibody solution at 37°C with shaking (2 days)
- Wash sample with buffer (1 day)
- Incubate sample in secondary antibody solution at 37°C with shaking (2 days)
- Wash sample with buffer (1 day)
- Note: Greater antibody concentrations and longer incubation times are needed for effective immunostaining. Starting with a 1:50 primary and secondary antibody dilution is recommended and can be adjusted as needed. The given incubation times work well 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 (Days 22-25)
- Incubate sample in FocusClear solution for several hours (or overnight) at room temperature 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: Do not store the samples in FocusClear. An irreversible white precipitate can develop inside the hydrogel after several days.
Step 9: Multiple staining rounds (if desired)
- After removing sample from FocusClear in imaging chamber, place in 50 mL PBST at room temperature 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 with shaking for 1-2 days to wash out the clearing solution
- Repeat Steps 7 and 8 with the next round of antibodies