Processing of a Mouse Brain for Downstream Analysis

Take a freshly harvested mouse brain and flash-freeze it in pre-chilled isopentane for rapid tissue preservation.     

Next, transfer the brain, cortex side up, into a frozen brain matrix to maintain a cold environment during slicing.

Allow the brain to equilibrate to the matrix temperature. Align it in the matrix along the sagittal and transverse sinuses to ensure symmetrical sections.

Insert chilled blades into the brain, ensuring proper alignment.

Then, press them down evenly to obtain uniform brain sections.

Remove the blades with the sections and position them, rostral side up, on a frozen glass plate.

Organize the sections from rostral to caudal orientation in the correct anatomical order. Now, separate them.

Using a suitable brain reference, locate and excise the region of interest from the section.

Transfer the excised tissue into a pre-chilled vial for downstream analysis.

After removing brains from euthanized adult CD-1 wild-type mice flash freeze the tissue for 60 seconds in either liquid nitrogen or isopentane prechilled with dry ice, and store it at -80 degrees Celsius.

24 hours before dissecting the tissue, place a clean brain matrix on a stack of thawed freezer packs, and sandwich the sides of the matrix between two freezer packs, making sure to leave approximately half a centimeter between the bottom of the razor slots and the top of the packs.

Set up a frozen glass plate for the dissection by filling an insulated box with ice up to approximately 5 centimeters from the top. Then, place a 2.5-centimeters layer of dry ice on top and cover it with black plastic sheeting. Place a glass plate on top of the plastic and dry ice around the borders of the plate.

Remove the frozen brain matrix from the freezer and insert the brain cortex side up into the matrix. Allow the tissue to equilibrate to the temperature in the box for 10 minutes, keeping the lid open during this time. Use cold forceps to adjust the brain's position in the matrix, so that the sagittal sinus and transverse sinus line up with the perpendicular grooves of the block.

Once the brain is in position, place a chilled razor blade near its center and press it approximately 1 millimeter into the tissue. Next, place one chilled blade at each end of the brain and press all the way down into the matrix. Then, begin adding chilled blades at the rostral end, placing them into the slots one at a time and gently pressing them 1 millimeter into the tissue. Continue to add blades at 1-millimeter intervals, working toward the caudal end.

When all blades are in place, press down on top with fingers, palm, or a blunt object, and rock them slowly from side to side to move them through the tissue. Once the blades have reached the bottom of the slots, grasp each side of the group of blades, and free them from the matrix by rocking back and forth.

After freeing the group of blades, place them rostral side up on the glass plate, and put dry ice next to or on top of the stack to further freeze the samples for easier separation. Then, place the stack with sharp edges down and separate the blades by shifting the stack between the thumb and fingers.

Line up the section on the glass plates from rostral to caudal, and separate the tissue from the blades by flexing them between the fingers or by separating them with a second chilled blade. At times, tissue may stick to both sides of a blade and care must be taken to maintain rostral-to-caudal orientation.

Before starting the dissection, open the Allen Mouse Brain Atlas or another reference and find the landmarks necessary to identify regions of interest. Use chilled forceps or blades to flip the section, and make sure that the region of interest is consistent throughout the section.

Cut into the section with a clean scalpel or punch, pushing the metal gently but firmly into the tissue, rocking it back and forth to make the cut. After harvesting the region of interest, place it into labeled prechilled 1.5-milliliter tubes, and store them at -80 degrees Celsius.