TITLE: Refractive Index Matching - EasyIndex
AUTHORS: Holly Myers, Daphne Toglia

[DESCRIPTION]
This protocol is modified from the Index Matching protocol found in the LifeCanvas Technologies Full Active Pipeline Protocol (see references). At the end of this process, the specimen should have the same refractive index as LifeCanvas EasyIndex, allowing for lightsheet imaging on the SmartSPIM. Refer to the following protocols to prepare for imaging: Embedding Protocol - SmartSPIM. EasyIndex Agarose Embedding Protocol, SmartSPIM setup and alignment, and Imaging cleared mouse brains on SmartSPIM.

[BEFORE_START]
This protocol is intended for a brain that has already been delipidated. See protocol Whole Mouse Brain Delipidation - LifeCanvas Active for preceding steps. 

To allow time for troubleshooting (step 5), it may be beneficial to begin this protocol at the start of day.

[STEPS]
SECTION: LifeCanvas Index Matching
1. Shake bottle of LifeCanvas EasyIndex well to homogenize the solution. Let the bottle sit for 30 min to allow any bubbles to settle. 
SECTION: LifeCanvas Index Matching
2. Using a serological pipet, create 20mL of 50% EasyIndex by measuring 10mL of nuclease free water and 10mL of LifeCanvas EasyIndex into a 50mL falcon tube. Place upright on shaker at low speed for 5 min to mix.

Add one delipidated whole mouse brain specimen into the tube and make sure specimen is completely submerged in solution. A long-handled spatula may be used to transfer the mouse brain into the tube. Seal tube tightly to prevent evaporation and cover in aluminum foil to protect from light. Incubate for 1440 min at 45 °C on shaker at slow speed.
 

SECTION: LifeCanvas Index Matching
3. Remove the 50% EasyIndex solution from the tube containing the specimen by using a long-handled spatula to scoop the brain out of the tube and pouring out the 50% EasyIndex solution from the tube. Replace the brain into the tube. Using a serological pipet, measure 20mL LifeCanvas EasyIndex into the tube and incubate for 1440 min in oven at 45 °C on shaker at slow speed.
SECTION: Assessing for Successful Index Matching
4. Remove tube with brain specimen and assess brain for successful index matching. The brain may be temporarily immersed in EasyIndex in a clear petri dish for evaluation.

 

Successful index matching:
When fully immersed in EasyIndex solution, the brain should be completely transparent. It should be possible to view numbers, letters, or fine lines on a grid through the brain without warping.

If the brain successfully index matched, proceed to step 6.


 


Not successfully index matched yet:
When fully immersed in EasyIndex solution, some opaque, white, or partially transparent structures may be visible inside the brain. It may be difficult to view images or lines through the brain without warping.

If the brain does not look successfully index matched yet, proceed to step 5. 

 

SECTION: Assessing for Successful Index Matching
5. Place tube with the EasyIndex solution and brain specimen back into the 45 °C oven on shaker at low speed for an additional 360 minand assess brain specimen for transparency again (step 4).

If brain specimen is not yet completely transparent, remove the EasyIndex solution from the tube and replace with 20mL fresh EasyIndex. Replace specimen in 45 °C oven for at least 960 min  on shaker at low speed.

This additional step should maximize index matching between the brain and the EasyIndex solution as much as possible. Proceed to step 6.

SECTION: Assessing for Successful Index Matching
6. The brain specimen is now ready for embedding, if desired. See protocol Whole Brain Embedding - EasyIndex with 2% Agarose for more details.

Sample may be stored in EasyIndex for several months at Room temperature .