This method can help answer key questions in the neuroscience field such as the analysis of the structure and the function of the neocortex. The main advantage of this technique is that it can reveal the large-scale three-dimensional structure in the brain. Though this method can provide insight into the neocortex, it can also be applied to other nervous systems such as the spinal cord.

To inject the tracer into the pons of the adult mice, first draw one microliter of fluorescently-labeled Cholera toxin subunit B into a 26-gauge Hamilton syringe. Place an injector pump on the syringe, then place the syringe and pump on the tool holder of a manipulator on a stereotaxic instrument. Tilt the manipulator 12 degrees posteriorly from the vertical axis.

Afterward, place the mouse on the stereotaxic instrument. Using a razor blade, remove its hair to prevent infection. Then cut 10 millimeters of the scalp so that the bregma and lambda are visible.

Administer 0.1 milliliters of 1%lidocaine using a pipette. Set the angle of the head by adjusting the vertical position of the mouthpiece on the stereotaxic instrument so that the bregma and lambda have the same Z level. Next, adjust the position of the manipulator by sliding it onto the stereotaxic instrument so that the tip of the syringe is close to the bregma.

Record the position of the manipulator. Retract the syringe by moving the tool holder on the manipulator. Subsequently move the manipulator 5.4 millimeters posteriorly and 0.4 millimeters laterally.

Advance the syringe so that the tip is close to the entry point on the skull, then retract the syringe and mark the entry point. At the marked position, drill a hole with a diameter of approximately one millimeter. Insert the syringe tip through the hole so that the tip depth is 6.9 millimeters more than that measured at the bregma.

Then, inject one microliter of tracers using the pump at 0.2 microliters per minute. After the injection, remove the syringe from the brain. If necessary, cover the exposed brain with small fragments of microfibrillar hemostat and instant adhesive.

Wipe the exposed brain with saline to prevent infection and suture the scalp. Next remove the mouse from the stereotaxic instrument. Allow the mouse to recover from anesthesia in an incubator at 30 degrees Celsius for about an hour.

Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency. Return the mouse to the company of other animals after it is fully recovered. To collect the brain tissue, cut the scalp using a pair of scissors to expose the skull.

Next, cut the midline of the exposed skull using a pair of scissors. Then, remove the skull using forceps. If marking the position of the bregma and lambda is necessary, first remove one side of the skull.

Then insert thin tungsten needles into the brain at the positions of the bregma and lambda, then remove the remaining skull. Subsequently, place the brain sample on the vibratome platform. Cut the sections up to 500 micrometers thick in PBS at room temperature.

In this procedure, transfer the slices to a five-milliliter plastic tube containing four milliliters of Scale S0 solution. Then incubate them for 12 hours with gentle shaking at 37 degrees Celsius. After 12 hours remove the solution from the tube using a pipette and add four milliliters of Scale A2 solution.

Incubate the slices for 36 hours with gentle shaking at 37 degrees Celsius. After 36 hours, remove the solution from the tube and add four milliliters of Scale B4 solution. Incubate the slices for 24 hours with gentle shaking at 37 degrees Celsius.

After 24 hours, remove the solution from the tube and add four milliliters of Scale A2 solution. Incubate the slices for 12 hours with gentle shaking at 37 degrees Celsius. After 12 hours, remove the solution from the tube and add four milliliters of PBS.

Incubate the slices for six hours with gentle shaking at room temperature. Subsequently, carefully remove the slices to a two-milliliter plastic tube. Incubate them with a primary antibody in one milliliter of AB Scale solution for 48 to 72 hours at 37 degrees Celsius with gentle shaking.

Afterward, carefully remove the slices to a five-milliliter plastic tube. Incubate them in four milliliters of AB Scale solution for two hours two times at room temperature with gentle shaking. Afterward, carefully remove the slices to a two-milliliter plastic tube.

Incubate with a fluorescently-labeled secondary antibody in one milliliter of AB Scale solution for 48 hours at 37 degrees Celsius with gentle shaking. Carefully remove the slices to a five-milliliter plastic tube containing four milliliters of the antibody Scale solution and incubate the slices for six hours with gentle shaking at room temperature. Remove the solution and add four milliliters of the AB Scale solution and incubate the slices for two hours two times with a gentle shaking at room temperature.

Next, remove the solution and add four milliliters of 4%PFA and incubate the slices for one hour with gentle shaking at room temperature. Then, remove the solution and add four milliliters of PBS and incubate the slices for one hour with gentle shaking at room temperature. Remove the solution and add four milliliters of the Scale S4 solution and incubate the slices for 12 hours with gentle shaking at 37 degrees Celsius.

Subsequently, place the spacer on a glass slide and place the slices in the spacer and immerse the slices in Scale S4 solution. Seal the spacer with a cover glass. Put some water on the cover glass and image the slices using confocal or two-photon microscopy with a water-immersion long-working-distance objective.

In this study, cortical projection neurons were labeled in green by tdTomato expression in Tlx3-cre AI9 transgenic mice and sub cerebral projection neurons in magenta were visualized by injecting the retrograde tracer CTB488 into the pons at seven weeks of age. This is the top view to show the approximate positions of cortical areas. Here are the oblique views of CTB488 fluorescence showing sub cerebral projection neurons and tdTomato fluorescence showing cortical projection neurons and this is the merged image.

The cell bodies of the two cell types were visible over a wide range of brain areas and optical sections showed periodic micro columns. This image shows the cell bodies and apical dendrites of eGFP-expressing sub cerebral projection neurons in optical section of a Crym-eGFP mouse and this image shows the parvalbumin-expressing cells labeled in green and sub cerebral projection neurons labeled in magenta by injecting CTB488 into the pons of a C57 Black 6 mouse by the antibody Scale method. Following this procedure, other methods like 3D image processing can be performed in order to answer additional questions like quantitative characterization of the cerebral organization.

After its development, this technique paved the way for researchers in the field of cortical biology to explore novel modular organization in the mouse brain.