Our research primarily focused on investigating the neuro circuits mechanism underlying connective impairments in mice and we frequently employed brain stereotaxic techniques in these studies. Our objective is to optimize and accelerate the verification process for stereotaxic injection sites using dyes, ensuring that it becomes a simple and rapid procedure. Virus strategies have become crucial in neuroscience research through stereotaxic injection of virus.

This is followed by observation of fluorescent markers in tissues lines to confirm the injection site, a process that takes several weeks to complete. Our protocol offers advantages over other techniques by saving us three days of dehydration time and using a cost-effective dye for quicker identification of injection sites. It may save weeks, compared with no preliminary verification.

Begin by placing the sterile surgical instruments on the working bench. To secure the anesthetized mouse in the stereotaxic frame, mix the upper incisors with a nose clamp and stabilize the head with two ear bars. Using eye scissors, make a 0.5-centimeter incision on the scalp to expose the cranium.

With a cotton swab dipped in 3%hydrogen peroxide, scrub the exposed skull to remove the periosteum. Allow the cranium to dry. Affix the drill to the stereotaxic instrument and adjust the stereotaxic arms'knobs.

With the help of a magnifier, position the drill tip precisely at the bregma. Record the Z-axis value at the bregma using the LCD digital display. Now position the drill tip at the lambda and record the Z-axis value.

Adjust the nose clamp to minimize the difference between the two Z values. Position the drill at coordinates minus 5.2 for X and plus 0.8 for Y to measure the Z-axis value in the dorsal ventral direction. Reposition the drill at coordinates minus 5.2 for X, minus 0.8 for Y, and measure the Z-axis value again.

Adjust the ear bars to a specified scale. Position the drill to the bregma and set three coordinates on the LCD digital display to zero. Using reference coordinates, position the drill above the region of interest.

Start the drill and gradually lower it using the vertical arm until the drill penetrates the cranium. Clear away debris and blood using cotton swabs. Add 25 microliters of SDS-PAGE loading buffer containing Bromophenol blue and 50 microliters of double-distilled water to a 200-microliter tube.

Cover the cranial window with a saline moistened cotton pad. Load 0.3 microliters of dye into the micro syringe. Attach the syringe and the motorized stereotaxic micro injector to the stereotaxic arm.

Position the syringe tip at the bregma and set the coordinates to zero on the LCD digital display. According to reference coordinates, adjust the needle into the region of interest. Set the injector speed to 0.1 microliter per minute and start the motorized micro injector, delivering 0.3 microliter of dye into the target region.

After 10 minutes, slowly withdraw the injector. To begin, release the Bromophenol blue dye-injected mouse from the stereotaxic frame. After anesthesia, secure the mouse on a foam board with tape.

Make a one-to two-millimeter incision in the right atrial appendage. Insert an infusion needle into the left ventricle. Infuse 20 milliliters of pre-cooled saline, followed by 20 milliliters of iced 4%paraformaldehyde solution.

Now make an incision along the midline from the neck to the nose to remove the skin and expose the skull. Use forceps or scissors to clear off residual muscle on the skull. Place the tip of one blade of the fine scissors between the foramen magnum and the brain with a sharp edge facing the bone at the rostral end of the mid sagittal suture.

Gently slide and cut the cranium along the mid sagittal suture. Use forceps to remove the skull, exposing the brain. After turning on the rotary microtome cryostat, set the chamber temperature to minus 20 degrees Celsius.

Pour a little optical cutting temperature or OCT compound to cover the surface of the specimen disc evenly. Place the specimen disc into the cryochamber and allow the OCT to solidify. Attach the disc to the specimen head.

Then bring the blade closer to the block and trim the OCT block to make a flat plane parallel to the disc. Using a shaving blade, cut the brain in a coronal direction away from the injection site in a brain slice mold. Place the brain tissue containing the injection site with the cut side down on the OCT flat plane.

Pour OCT onto the brain and place the disc into the cryochamber, allowing the specimen block to solidify. Trim the specimen block until the target region is approaching and make several sections from the injection site level. Use a chilled small writing brush to collect the coronal brain slices into a six-well plate containing room temperature PBS.

Using a brush, pick up the brain sections from the six-well plate and place them on a slide. After drawing, visually observe the injected region in the brain sections.