Brain Block One™ Concept
Figure 1. Brain Block One for Mice Series of 4 parts (blue food coloring dye used to improve contrast for photography)
EMS Catalog # 69010
Whenever laboratory animal brain tissue is to be thin sectioned for microscopic examination, alignment of the organ in recognizable planes is always an issue. Consistent alignment in planes found in reference atlases greatly facilitates locating and identifying structures or artifacts in the brain when sections are examined under the microscope, and in comparing between brains.
Cryostats and microtomes have mechanical mechanisms for adjusting orientation after cutting a few sections in the starting plane. The process is trial and error, slow, and requires wasting several sections after each adjustment.
Matrices are metal or plastic blocks with cavities in roughly the shape of a brain, and slots to guide a blade. These are used to make a starting cut, approximately in a consistent plane, to reduce the need for adjustment in the cutting stage. The brain can move about a bit, the blade can stick to the tissue and push movement. The cut is very approximate, seldom exactly consistent.
The Brain Block One™ encasement gels are right and left side rectangular blocks. A cavity in each gel block was originally cast around an actual adult brain in skull flat alignment. The skull flat plane is thus perpendicular to the ends, and parallel to the outer surface, of each block. An exacting procedure was used to make the cavity perfectly aligned with the block edges.
A brain may be precisely and immovably encased in the gels to look like it was embedded there, and the entire block frozen and sectioned, putting any surface down on the cryostat or microtome pedestal to ensure precise orientation in the desired plane.
Encasement gels are available for mice and rats, check to see it we have made them for the species you work with.
The gels will come packaged in individual pairs. They are slightly dehydrated and therefore slightly shrunken when you unpackage them. To use for fresh adult brain, soak for 1-4 hours in saline, PBS, or your chosen water based media.
After soaking, they will work as illustrated in the photo series of Figure 1, to the left. The first box shows a vacant right side gel cavity.
The next box shows a fresh mouse brain dissected out and dropped directly into a presoaked gel.
The third picture shows the gel with the top placed on to fully encase the brain. Note that the line around the center is nearly invisible; the gel has closed around the brain and leaves no seams or air pockets.
The last picture of Figure 1 shows the block in another orientation, top down, and illustrates that this brain is in skull flat orientation within the gel, parallel to the present bottom side of the gel block. The blocks can be placed in any orientation for sectioning in any desired plane, coronal, sagittal, or horizontal. They can also be placed in special planes for which the pedestal has been preset to a preplanned angle.
Multiple Brain Sectioning
As another useful option, multiple blocks containing brains may be positioned together side by side or end to end, and each pass of the knife can take the same section from each brain. Collection of these types of sections, and transfer to 1 x 3 or 2 x 3 slides (holds up to 24 mouse brain sections) can be facilitated by a tape transfer system.
Figure 2. Dry, 4 hour, and 24 hour soaked gels showing range of expansion.
Figure 3. Gel too shrunk from dehydration to close
Gels will continue to expand in water for 24 hours or more. Figure 2 shows a gel just removed from the package, an identical gel after soaking for 4 hours, and another identical gel soaked for 24 hours. If not soaked, they may not close about the animals brain when inserted. Figure 3 illustrates this problem.
If the gels are soaked a little longer than necessary, and the cavities are slightly larger than the brain, capillary action will retain a thin layer of saline or media in the gap around the brain, and this will freeze and section. This is acceptable. If the cavities are larger still, capillary action cannot hold on to the fluid, and it will drain out the seam between halves and leave an air gap. This is unacceptable. There is about a 2 hour range of perfect, and an hour or more of acceptable, soak time.
Cavity Size Adjustment
The ability to adjust the size of the cavity by soak time will prove useful for brains of young animals, poorly perfusion fixed and therefore shrunken brains, and for brains of older animals. The range of acceptable soak times, about 3-4 hours, gives ample working space.
- Put paired gel halves on to soak, from 1-4 hours for fresh adult animals of atlas age. Space out the beginning of soak time if sacrifice will take time.
- Sacrifice the animals, extract the brain, and drop it into a fluid filled gel half in the orientation of the gel.
- Lift that gel with the brain sitting in it, tip it into the mating gel so the brain fits into the cavity of the next gel half. The brain should always displace fluid as it fits into the cavity, so the cavity being filled should be facing up.
- When the gel halves fully encase the brain, the block can be moved onto a plastic weigh boat, and moved onto a freezing plate. Many methods may work, including immersion freezing in isopentane/dry ice slurry, but we have used a ¾ inch thick aluminum plate, left sitting on a pool of dry ice and alcohol, so the alcohol is in contact with the plate, all in a large plastic pan with a cover over it. Let this equilibrate, then set a weigh boat with gel and brain on the plate, cover the whole thing, and let it freeze. Keep covered to minimize frost buildup. See Figure 4. This procedure may seem likely to cause freezing artifact due to slow freezing, but it does not. Apparently, each layer freezes abruptly.
- Finished and frozen blocks may be cemented together with OCT and cemented to a pedestal for sectioning together. Use the reference faces to align.
These gels are a new product. New applications are possible. It is also conceivable that will be problems that have not been encountered. Please contact us for assistance.