Extraction and Tissue Clearing Preparation of Mouse Brain-Spinal Cord Samples

Our research focuses on how the central nervous system mediates the cognitive functions, and interacts with the immune system. We aim to improve the methods for examining the brain and the spinal cord, addressing the fundamental questions about these interactions. These methods keep the whole brain and the spinal cord intact, which help us map the cell more thoroughly using tissue clearing. It also makes the process quicker and more accurate, reducing time and the mistakes compared to the traditional sectioning and imaging methods.

We have mapped the brain and the spinal cord regions connected to the lungs yielding . Our research will focus on exploring the neuro immune communication between the brain, spinal cord, and the lungs. And how this interaction influences immune responses.

[Instructor] To begin, fix the mouse's limbs to the wide acrylic board using steel clamps. Using scissors, cut the skin between the two femurs and continue cutting upwards until reaching the diaphragm. Open both sides of the thorax through the diaphragm, using scissors and straight forceps to expose the heart. Insert a 0.7 millimeter infusion needle attached to a perfusion tube into the left ventricle, and make a one to two millimeter incision in the right atrial appendage. After perfusion, separate the skin on the back and head of the mouse using ophthalmic scissors. Sever the spine at the point where the line connecting the upper rear edges of both limbs intersects with the vertebral column. Remove muscle and fat tissue from the neck and back of the mouse to expose the spine and skull. With venous scissors, cut along the midline gap between the left and right sides of the spine and spinal cord from the lower end of the vertebral canal. Then remove the upper portion and bilateral sides of the severed spine to expose the spinal cord. Repeat the steps until reaching the brain connection. Place the tip of one blade of the venous scissors between the foramen magnum and the brain. Slide and cut along the midsagittal suture to open the skull. Use forceps to remove the skull and expose the brain. Now, using forceps and venous scissors, remove the mouse brain spinal cord tissue from the cranial end. Use dissecting forceps to remove the spinal cord membrane under the stereo microscope. Next, with sutures, secure the brain spinal cord sample to the fixing plate. Add at least 20 times the sample volume of 4% paraformaldehyde. After fixation, discard the paraformaldehyde and wash the sample three times with PBS. To begin, fix the extracted mouse brain spinal cord in paraformaldehyde. For the lipid removal solution, mix solution A and B at a mass ratio of nine to one. Add 50 milliliters of the lipid removal solution to a 50 milliliter centrifuge tube and place the fixed tissue in it. After incubation in solution C, change the solution and place the culture dish with the sample fully immersed in solution C on a light box with a scale line. To prepare gel solution, add 147 grams of solution C and three grams of agarose to a 200 milliliter glass bottle. Mix the components well with a stirring rod, then microwave until the agarose dissolves. Transfer the gel solution to a laboratory oven set to 37 degrees Celsius. Pour a two millimeter deep layer of 37 degrees Celsius tempered gel solution into the mold, and cool it in a four degrees Celsius refrigerator for 30 minutes until it is semi-solid. Place the refractive index matched sample into the mold, and add gel solution up to the level or slightly below the top of the sample. Place the mold back in the four degrees Celsius refrigerator for two hours. Cover with a cover slip and refrigerate again. Remove the cover slip with tweezers and take out the embedded sample. Add 30 milliliters of imaging solution to a 50 milliliter tube and submerge the sample in the solution. Apply 502 adhesive to firmly affix the sample onto the sample holder, before fixing the holder onto the imaging device. Run the scanning software and select the 4X magnification level in the magnification section. Click the calibration button to activate the calibration mode. Click the preview button to enter preview mode. Then click the Z direction movement button to slowly move the sample upward from the bottom of the sample chamber until the generated light sheet passes through the sample. Select the appropriate laser channel and illumination mode option. Select the scanning path direction for the sample. Click the auto scan button to start capturing the images of the sample. The protocol successfully preserved the entire mouse brain and spinal cord while maintaining intact fluorescent signals for clear imaging. The fluorescent imaging of the brain spinal cord sample clearly visualized detailed neural structures. Higher magnification images provided enhanced visualization of individual fluorescently labeled cells.