Collection of Frozen Rodent Brain Regions for Downstream Analyses

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools. Because brain regions are kept frozen from harvest through dissection, target molecules are preserved and the researcher has time to carefully dissect and store regions of interest. Identifying regions of interest can initially be challenging.

Using common brain landmarks as they emerge and recede through the sections in relation to the desired regions will make identification clear. After removing brains from euthanized adult CD-1 wild type mice, flash freeze the tissue for 60 seconds in either liquid nitrogen or isopentane. Pre-chill with dry ice and store it at minus 80 degree Celsius.

24 hours before dissecting the tissue, place a clean brain matrix on a stack of thawed freezer packs. And sandwich the sides to 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 five centimeters from the top.

Then place a 2.5 centimeter 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 one 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 one millimeter into the tissue. Continue to add blades at one 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 a 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 pre-chilled 1.5 millimeter tubes and store them at minus 80 degree Celsius.

To process the tissue, add cold RIPA buffer for protein extraction or a guanidinium containing solvent for RNA extraction and immediately homogenize it with a glass downs or mechanical homogenizer. To validate this method, the medial prefrontal cortex was collected from adult CD-1 wild type male mice, and RNA and protein were characterized. When analyzed with capillary electrophoresis, degraded RNA displays a loss in the intensity of the 28S and 18S ribosomal bands, as well as a smear between 25 and 200 nucleotides.

While high-quality RNA shows distinct ribosomal bands little to no signal in the lower molecular weight region. The RNA obtain using the frozen dissection method was compared with RNA prepared from freshly harvested tissue. Both methods produce RNA with high integrity numbers and strong ribosomal banding.

To confirm that this method can be used to preserve the microenvironment of dissected tissue for later analysis, RNA was extracted from dissected medial prefrontal cortex that had been stored over several weeks. All samples produced high-quality RNA with distinct ribosomal banding and RNA integrity numbers above eight. To confirm protein integrity of the frozen dissected samples, the protein was collected, transferred to nitrocellulose and probe with antibody against the high molecular weight target KCC2, and the lower molecular weight protein actin.

In all cases bands were sharp and distinct with no discernible breakdown products. It is important to keep the tissues frozen throughout the process as this preserves the microenvironment of the sections and maintain section morphology for comparison with brain atlas images. Regions of interest collected in this manner can be stored for several months at negative 80 degree Celsius and can be utilized in many downstream applications, including RT-qPCR, RNA-Seq, Western blot analysis, and HPLC.

This method has been used to explore molecular changes within the limbic systems of perinatal rodents exposed to THC and other cannabinoids to better understand how these drugs may affect brain development.