This time-saving method reduces variability between rounds of immunohistochemical procedures in neuroscience studies to optimize the visualization of anatomy and local proteins and tissue. The main advantage of this technique is that it reduces the time taken to cryosection and mount brain tissue by combining multiple brains into a single frozen block. While this technique is optimized for coronal sections of rodent brains, it can be adapted to sagittal or transverse sections or different types of tissues such as muscle or liver.
Following successful transcardial perfusion, post-fix the brains from the animals in a 25 milliliter vial of 4%paraformaldehyde in PBS for 24 hours. After this, use blunt tipped forceps to remove the brains from the paraformaldehyde. Transfer the brains to a vial with approximately 20 milliliters of 30%sucrose solution in TBS.
Keep the brains and solution until they sink to the bottom of the vial. Next, place a 500 milliliter glass beaker on a bed of dry ice in an ice bucket. Then pour 300 to 400 milliliters of isopentane into the beaker.
Maintain the isopentane between negative 45 and negative 50 degrees Celsius. On the bench top, fill a disposable embedding mold approximately halfway full with Optimal Cutting Temperature or OCT compound. Use a needle to remove any air bubbles from the mold.
Use the blunt tipped forceps to remove the first brain from the sucrose solution. Then use a new razor blade to remove the cerebellum and olfactory bulbs before separating the hemispheres. Next, give the brains a numeric naming system.
Use blunt forceps, pick up the brain to be placed in position two, and orient it with the side to be cut first facing upwards. Lower the brain into the OCT and use the tweezers to adjust its position until it stands independently. A key modification of this technique is the blank space in position one.
This space is designated to allow easy identification of the megabrain orientation and therefore identify the animal associated with each brain. After all of the brains have been positioned in the mold, add sufficient OCT to cover the tops of the brains, then use a needle to remove any air bubbles. Hold the corner of the mold with forceps ensuring the forceps do not become partially submerged in the OCT.
Then, lower the mold into the isopentane so that the bottom third of the mold is submerged. After 30 seconds, lower the entire mold into the isopentane and release it from the forceps. After three minutes, use the forceps to remove the mold from the isopentane.
Immediately wrap the mold and its contents in aluminum foil and label it appropriately. Transfer the megabrain to a negative 20 degree Celsius freezer for a minimum of 24 hours. First, set the temperature of the cryostat cabinet to negative 19 degrees Celsius.
Remove the megabrain from the freezer and place it in the cryostat. Then place a chuck and two thin tipped paintbrushes in the cryostat. After this, label the slides with the megabrain identification number and slide number and lay the slides on the slide warmer.
Next, unwrap the megabrain and use a razor blade to cut the corners of the mold. Dispense a thin layer of OCT onto the chuck. Then quickly place the megabrain on the chuck.
After the OCT is completely frozen, apply a two millimeter layer of OCT around the sides of the megabrain and allow it to run down the sides onto the chuck. Use a razor blade to remove any excess OCT from the sides and bottom of the chuck. First, position the chuck mounted with the megabrain on the chuck head of the cryostat.
Then set the cryostat to the desired thickness. Trim the OCT and tissue as needed to reach the desired brain region for tissue collection. Then lower the anti-roll plate until it rests on the stage and turn the cryostat handle to take a single section.
Slowly lift off the anti-roll plate, being careful that the tissue section is not touched. Gently use the paintbrushes to unroll the tissue section until it's lying flat. If needed, hold the OCT flat using the paintbrushes to prevent rolling.
Then remove the slide from the slide warmer and hover the slide label side down over the megabrain section and allow it to stick to the slide. Quickly apply a drop of PBS to the tissue section. Using a fine tipped paintbrush dipped in PBS, brush out any bubbles in the tissue section and unfold the tissue so it is lying flat on the slide.
Finally, place the slide on the slide warmer to dry for 45 minutes and store the slide at negative 80 degrees Celsius. In this protocol, brain tissue from nine animals were prepared and cryosectioned simultaneously. Following cryosectioning, H&E staining can be carried out to assess the cryoprotection quality.
Representative results of ionized calcium binding adapter molecule one or Iba1 staining of microglia show consistent staining between each study brain on a single megabrain. While attempting this procedure, it's important to remember to constantly monitor the temperature while freezing the megabrain to prevent cracking or thawing and then refreezing of the tissue which leads to abnormal tissue integrity. It is not advised to segregate different treatment groups in separate megabrains as this can create variance between groups during staining and reduce bias and subjectivity during imaging and analysis.
Don't forget that working with paraformaldehyde can be extremely hazardous and precautions such as wearing PBE and working in a hood should always be taken while performing this procedure.
