Preparation of Colliculo-Thalamocortical Slices from a Mouse Pup Brain
Transcrição
To prepare the cutting stage for slicing, cut a small piece of 3% agar of approximately 1 cubic centimeter to be used as a backstop for the brain, and another piece of 1.5 centimeter by 1.5 millimeter by 3 millimeters to be used as a bump to support the inferior colliculus.
Next, glue the backstop onto the stage with cyanoacrylate adhesive, and then glue the bump on the right side of the backstop such that they form an 80-degree angle. On a slide marked with two lines at 90 degrees and a diagonal line at 17 degrees from the top left to the bottom right, place the brain dorsal side up at the intersecting point of the two lines.
Using a razor blade, remove 2 to 4 millimeters of the rostral end of the brain to create a flat surface. Next, place the brain caudal side up on the newly created flat surface. Align the dorsal surface of the brain with a horizontal line, and the midline of the brain with a vertical line marked on the slide. Then, align the razor blade with a 17-degree line. Tilt the razor at a 30-degree angle, and remove approximately 3 millimeters of the right cortex in a double diagonal cut.
To mount the brain on the vibratome stage, place a small piece of filter paper on the ventral side of the brain so that the long dimension is perpendicular to the midline. Carefully apply a small amount of cyanoacrylate adhesive to the area in front of the backstop and to the left of the bump. Subsequently, place the double diagonally cut brain side onto the glue so that the caudal part of the brain and the hindbrain are propped up onto the bump, and the right side of the brain is against the backstop.
In this procedure, quickly place the cutting stage in the vibratome. Fill the stage with cutting solution. Next, align the blade with the top of the brain. Remove 1 to 1.5 millimeters from the top of the brain. Then, slice and remove more slices of 300 to 500 micrometers while moving deeper.
When the inferior colliculus, the medial geniculate body, and the lateral geniculate nucleus are all visible, the dentate gyrus should appear as a C-shape, and the structure should be in alignment. Obtain about two slices of 600 micrometers. These are the colliculothalamocortical slices. After that, transfer them to the 32-degree Celsius holding chamber.
