CLARITY Timeline

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(Step 4: Clearing solution wash (2 days minimum))
(Step 4: Clearing solution wash (2 days minimum))
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*Replace 50 mL clearing solution and continue incubation at 37°C or room temperature
 
*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.
 
**''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 washes as desired or store in clearing solution. The tissue will clear slowly from passive diffusion of SDS micelles.
+
*Repeat clearing solution washing as desired or store in clearing solution. The tissue will clear slowly from passive diffusion of SDS micelles.
 
**''Note:'' Samples can be stored indefinitely in the clearing solution.
 
**''Note:'' Samples can be stored indefinitely in the clearing solution.
   

Revision as of 23:00, 31 March 2014

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.

Contents

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)

  • De-gas the sample container to remove oxygen from inhibiting polymerization
  • Incubate the sample at 37°C for about 3 hours to polymerize and crosslink the hydrogel matrix
  • 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

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: Samples can be stored indefinitely in the clearing solution.

Step 5: Electrophoretic tissue clearing (ETC) (~3-6 days)

  • 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 6 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 (2 days minimum)

  • 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 (6 days for 1 mm thick slices)

  • 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 (0-3 days)

  • 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