CLARITY Timeline
From CLARITY Wiki
(Difference between revisions)
(→Step 3: Hydrogel tissue embedding (1 day)) |
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*Make or thaw the [[Solutions#Hydrogel Solution|hydrogel monomer solution]] |
*Make or thaw the [[Solutions#Hydrogel Solution|hydrogel monomer solution]] |
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*Complete [[Perfusion#Transcardial Perfusion|transcardial perfusion]] with the hydrogel solution (if possible) |
*Complete [[Perfusion#Transcardial Perfusion|transcardial perfusion]] with the hydrogel solution (if possible) |
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− | *Incubate sample in hydrogel solution at 4°C to let the hydrogel monomers diffuse inside the tissue |
+ | *Place sample in hydrogel solution at 4°C |
===Step 2: Hydrogel solution incubation (2-3 days)=== |
===Step 2: Hydrogel solution incubation (2-3 days)=== |
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− | *Continue sample incubation at 4°C |
+ | *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. |
**''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. |
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*Prepare [[Solutions#Clearing Solution|clearing solution]] |
*Prepare [[Solutions#Clearing Solution|clearing solution]] |
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*Remove excess gel from the sample surface (unless leaving out bis-acrylamide from the hydrogel solution) |
*Remove excess gel from the sample surface (unless leaving out bis-acrylamide from the hydrogel solution) |
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**''Note:'' If sectioning the sample, it is best to complete this immediately after hydrogel embedding before placing the sample in clearing solution. |
**''Note:'' If sectioning the sample, it is best to complete this immediately after hydrogel embedding before placing the sample in clearing solution. |
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− | *Place sample in 50 mL of clearing solution to wash out excess unreacted monomers from the tissue (37°C or room temperature, shaking preferable) |
+ | *Place sample in 50 mL of clearing solution |
− | ===Step 4: Clearing solution wash (Day 4)=== |
+ | ===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) |
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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 |
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**''Note:'' Samples can be stored indefinitely in the clearing solution. The tissue will clear slowly from passive diffusion of SDS. |
**''Note:'' Samples can be stored indefinitely in the clearing solution. The tissue will clear slowly from passive diffusion of SDS. |
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− | ===Step 5: Electrophoretic tissue clearing (ETC) (Days 5-14)=== |
+ | ===Step 5: Electrophoretic tissue clearing (ETC) (~3-6 days)=== |
*Set up electrophoretic tissue clearing (ETC) |
*Set up electrophoretic tissue clearing (ETC) |
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*Add sample to the ETC chamber and turn on clearing solution circulation and power supply |
*Add sample to the ETC chamber and turn on clearing solution circulation and power supply |
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*Routinely check the clearing solution pH and replace with fresh solution if it starts to dip below ~7.5 |
*Routinely check the clearing solution pH and replace with fresh solution if it starts to dip below ~7.5 |
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*Visually check the sample for clearing |
*Visually check the sample for clearing |
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− | **''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. |
+ | **''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 (Days 14-16)=== |
+ | ===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) |
*Remove sample from the ETC chamber when it appears see-through (sample will be swollen to a larger size and not visually transparent yet) |
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*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 |
*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 |
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**''Note:'' Samples can be stored indefinitely in PBST buffer. Store samples at room temperature or 4°C. |
**''Note:'' Samples can be stored indefinitely in PBST buffer. Store samples at room temperature or 4°C. |
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− | ===Step 7: Immunostaining (if necessary) (Days 16-21)=== |
+ | ===Step 7: Immunostaining (6 days for 1 mm thick slices)=== |
*Incubate sample in primary antibody solution at 37°C with shaking (2 days) |
*Incubate sample in primary antibody solution at 37°C with shaking (2 days) |
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*Wash sample with buffer (1 day) |
*Wash sample with buffer (1 day) |
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**''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). |
**''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). |
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− | ===Step 8: Sample imaging (Days 22-25)=== |
+ | ===Step 8: Sample imaging (0-3 days)=== |
*Incubate sample in FocusClear solution for several hours (or overnight) at room temperature with shaking |
*Incubate sample in FocusClear solution for several hours (or overnight) at room temperature with shaking |
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**''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. |
**''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. |
Revision as of 02:01, 6 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.
Step 1: Hydrogel solution and sample preparation (<1 day)
- Make or thaw the hydrogel monomer solution
- Complete transcardial perfusion with the hydrogel solution (if possible)
- Place sample in hydrogel solution at 4°C
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
- 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
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: 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) (~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