TITLE: Whole Brain Embedding for SmartSPIM - EasyIndex with 2% Agarose
AUTHORS: Holly Myers, Daphne Toglia

[DESCRIPTION]
This modified LifeCanvas embedding protocol is intended to prepare cleared mouse brains for imaging on the SmartSPIM (see Imaging cleared mouse brains on SmartSPIM). It details mixing and degassing the agarose solution and the technical process of embedding a brain in the agarose solution. 

Embedding the brain specimen in a solution of EasyIndex with 2% agarose before SmartSPIM imaging allows the brain to be suspended in the center of a block of index matching solution. The agarose block is glued to the SmartSPIM stand during imaging, sparing any area of the brain from being cut out of imaging. However, any imperfections in the agarose block, like air bubbles, may affect the image.

[BEFORE_START]
This protocol is designed for a whole mouse brain that has already been delipidated and index matched using these protocols: Editing Whole Mouse Brain Delipidation - LifeCanvas Active and Refractive Index Matching - EasyIndex.

2% agarose solution should be made at least one day in advance of embedding in order to fully hydrate the agarose powder in EasyIndex.

[STEPS]
SECTION: Setup
2. Preheat the water bath to 90 °C  and preheat the vacuum oven to 80 °C Preheating typically takes 60 min .

SECTION: De-gas agarose solution in vacuum oven
5.  Remove glass vial of homogenized agarose solution from water bath, uncap vial, and place in the vacuum oven preheated to 80 °C 

 

6. De-gas the agarose solution using the vacuum oven.

SECTION: Embedding
10. Remove vial of agarose from the vacuum oven and let the vial cool at Room temperature until warm but not hot to the touch, about 60 °C.

 

SECTION: Embedding
8. This step uses previously cleared adult mouse brain specimens (reference protocols Whole Mouse Brain Delipidation - LifeCanvas Active and Refractive Index Matching - EasyIndex). After following these protocols, the cleared brain specimens will be immersed in 20 mL EasyIndex solution in 50 mLconical tubes.

Remove the whole brain specimen from EasyIndex solution with a metal spatula and place brain onto a piece of lens paper, dorsal side down. Allow the brain to rest for at least 30 s to drain excess EasyIndex.

Retain the conical tube containing EasyIndex solution, storing at Room temperature .

 


SECTION: Embedding
11. Uncap the vial and align the mouth of the vial to the corner of one of the eight chambers of the embedding mold while pouring out the agarose solution at an even rate into the chamber. Pour until the embedding chamber is about 1/3 full of agarose solution. Cap vial and place back into the vacuum oven.


 

SECTION: Post-embedding
16. Place embedding block in 4 °C  fridge and ensure that block is covered to protect from light. A large plastic petri dish wrapped in aluminum foil may be used as cover for light protection. Leave 960 min. 

SECTION: Post-embedding
17. Use the black mold cutter to remove the agarose block containing the brain specimen.

SECTION: Prepare Agarose Solution
1. 5mL of 2% agarose solution is required for embedding a single brain specimen. Scale up as needed. Mix the solution in 20 mL glass scintillation vials.

 

 

SECTION: Set up Embedding Mold
4. Assemble an embedding mold secured to a breadboard that will be used to embed delipidated mouse brains in agarose blocks. After assembly, each chamber in the mold can hold a single brain, which can then be filled with molten agarose solution. After the agarose solution hardens, the mold will be disassembled to remove the hardened agarose blocks containing the brains.
SECTION: Set up Embedding Mold
4.1. Tape two glass slides into the two grooves in the plastic embedding mold. 

Note the four holes in the corners of the embedding mold and use these to determine which side of the mold to fasten the slides. On one side of the mold, the holes are surrounded by an indentation to accommodate the head of the screw, and the other side the holes are flush with the mold (see photo examples below). The slides should be taped to the side of the mold where the holes are flush and have no indentation.

 


 


 

SECTION: Set up Embedding Mold
4.2. Orient the embedding mold so that the side with the glass slides is flat down against the breadboard, and the side with the indentations around the four holes is facing up. The indentations around the holes will accommodate the heads of the screws.

 

7. Degassing is successful if there are no visible bubbles inside the agarose solution. Recap vial and leave vial in the 80 °C vacuum oven with door closed and vacuum off.

If air bubbles are still present inside agarose solution, repeat the degassing process from step 6.

 

SECTION: Embedding
15. Remove any small bubbles that form in the agarose solution in the mold with a P-200 manual pipette. Keep the brain centered in the embedding chamber as the agarose hardens.
SECTION: Embedding
14. Immediately after placing the brain into the embedding chamber, remove the vial of agarose from the vacuum oven and pour a second layer of agarose solution into the embedding chamber. This layer of agarose should totally cover the ventral surface of the brain. 

Stop pouring when the agarose is level with the top edge of the embedding chamber. The surface of the agarose solution should form a flat surface flush with the top surface of the mold. (not convex or concave).

 


 


 

SECTION: Post-embedding
18. Using gloved hand, gently place the agarose block into the conical tube containing 20 mL of EasyIndex solution that was set aside in step 11. Place tube on a shaker at low speed and let sit at least 960 min  at Room temperature before it is ready for imaging. Brains may be stored for up to several months at Room temperature in 20 mL of EasyIndex.

SECTION: Post-embedding
17.1. Unscrew the plastic embedding mold from the breadboard with a Phillips head screwdriver and gently remove the glass slides that are taped to the back of the mold.

SECTION: Post-embedding
17.2. Remove the agarose block with the brain embedded inside from the plastic embedding mold using the following directions:

Place the black plastic mold plunger on lab bench and lift the plastic embedding mold so that it is aligned a few inches directly above the black mold plunger. Slowly lower the plastic embedding mold down onto the mold plunger so that the protrusions on the mold plunger fit inside the holes in the embedding mold. Once the embedding mold is completely lowered down onto the mold plunger, the protrusions on the mold plunger will push the agarose block out of the embedding mold.

 





 


 


 

SECTION: Prepare Agarose Solution
3. Remove the glass container of agarose solution from the oven and aliquot 5 mL of agarose solution into glass vials with a serological pipet. Place glass vials containing agarose solution into preheated   90 °C water bath for 30 min or until agarose solution has thoroughly melted and homogenized. Homogenized agarose solution should be clear with the consistency of water.

SECTION: Set up Embedding Mold
4.3. Screw four (1/2") Phillips head screws through the four holes in the embedding mold to fasten the mold to the breadboard using a Phillips head screwdriver. This completes the assembly of the embedding mold.


 

SECTION: Embedding
13. After 80 s gently place transfer the brain specimen from the lens paper into the embedding chamber on top of the agarose solution, using either gloved hand or spatula. Keep the brain oriented dorsal side down, so the dorsal surface of the brain is touching the agarose in the chamber. Take care to set the brain gently on top of the agarose solution and not to push it down. The brain should slightly sink into the agarose but ideally should not sink through all of the agarose.

 


 

SECTION: Embedding
9. Use a marker to label one or more chambers in the embedding mold with the letter(s) that corresponds to the sample ID(s) of the brain specimen(s) to be embedded (reference protocol Whole Mouse Brain Delipidation- LifeCanvas step 3.2). This will prevent mix-ups between brain specimens as they are transferred from the conical tube to the embedding mold and back again.

 

SECTION: Embedding
12. Remove any small bubbles that form in the agarose solution in the embedding chamber with a P-200 manual pipette.
SECTION: Prepare Agarose Solution
1.1. Measure 0.1 g x number of brains of agarose powder on scale and place into a glass vial. Each glass vial should contain no more than0.2 g agarose; use multiple vials if needed.
SECTION: Prepare Agarose Solution
1.2. Using serological pipet, transfer (5 mL x number of brains) of EasyIndex into the glass vial(s) containing the agarose power. Each glass vial should contain 5 mL of EasyIndex for every 0.1 g agarose powder.

SECTION: Prepare Agarose Solution
1.3. Gently swirl the glass vial of agarose solution to pre-mix, and place vial on shaker at low speed in a45 °C oven. If this temperature oven is not available, 37 °C oven is sufficient. Leave  .
6.1. Turn on the "evacuate" setting on the vacuum oven to start the vacuum pump and begin de-gassing the agarose solution. 
6.2. When the solution starts to bubble, switch the vacuum oven setting to "stop/closed" to shut off the vacuum pump. This will happen once the pressure reaches around . Allow the agarose solution to bubble for 2 min.

 

6.3. Finally, switch the vacuum oven setting to "vent" to bring the vacuum oven back up to atmospheric pressure.