PCH Mission Support Funds supported the research reported in this manuscript. The authors would like to thank Daniel Griffiths for taking the images used in the figures.

The Megabrain technique has been optimized using whole and hemisected brains from male adult C57Bl6 mice5, juvenile Sprague-Dawley rats, and adult Sprague-Dawley rats that were transcardially perfused with 1x phosphate buffered saline (PBS) followed by 4% paraformaldehyde6. Similar outcomes can be accomplished in other strains of mouse and rat, in both sexes and at different ages. The study to generate data for this manuscript used juvenile Sprague-Dawley rats and was approved by the University of Arizona Institutional Animal Care and Use Committee, and experimental animals were cared for according the Guide For the Care and Use of Laboratory Animals7.
1. Cryoprotection of Perfused Brains
<ol>
	<li>Following successful transcardial perfusion6, collect the full brain6,8 from animals and post-fix by placing into a 25 mL vial of 4% paraformaldehyde in PBS for 24 h. 
	CAUTION: Paraformaldehyde is a toxic tissue fixative; handle with care. Transfer paraformaldehyde into a specifically labeled waste container that identifies its concentration and volume inside a chemical fume hood and then store in the chemical waste cabinet until disposed by Chemical Safety or properly trained personnel.</li>
	<li>After 24 h has elapsed, in a fume hood, remove brains from 4% paraformaldehyde using a spatula. Transfer the brain into a vial of approximately 20 mL of 15% sucrose solution in 1x Tris-buffered saline (TBS) until brain sinks to the bottom of the vial (approximately 24 h).</li>
	<li>Following this, transfer the brain to a vial of approximately 20 mL of 30% sucrose solution in 1x TBS, until the brain sinks to the bottom of the vial (approximately 24&#8211;48 h).</li>
</ol>
2. Brain Freezing
<ol>
	<li>Place a 500 mL glass beaker on a bed of dry ice in an ice bucket. Pour 300&#8211;400 mL of isopentane (2 methyl-butane) into the glass beaker.</li>
	<li>Allow the temperature of the isopentane to reach between -45&#160;&#176;C and -50&#160;&#176;C. Closely monitor and maintain this temperature range with a thermometer, keeping it constant throughout the procedure. Ensure that temperature readings are taken from the middle of the solution, and not while touching the bottom or side of the beaker.</li>
	<li>On the benchtop, fill a disposable embedding mold (see materials list) approximately 1/2 full of Optimal Cutting Temperature (OCT; see Table of Materials) Compound. Ensure that there are no air bubbles in the OCT. If there are any air bubbles remove them with a spatula or needle.</li>
	<li>Remove the first brain from the vial of 30% sucrose with a spatula. At this point, either freeze brains whole or hemisect, depending on the study design.
	<ol>
		<li>For hemisected brains, use a new razor blade to make a clean cut through the tissue to separate the hemispheres. If not required for the study, remove the cerebellum and olfactory bulbs at this stage. If the cerebellum is needed for the study, it is suggested to cut a flat area at the posterior end of the brain to aid the brain in standing straight in the mold. If the study only requires tissue from a localized region, a rodent brain matrix can be used to block the brain based on known coordinates.</li>
	</ol>
	</li>
	<li>Give brains a numeric naming system. Draw a diagram as shown in Figure 1. Write the number of the first brain to be placed in the Megabrain mold in position 2, leaving position 1 as a blank space to aid in quick identification of the orientation in which the brains were frozen.</li>
	<li>Using blunt forceps or tweezers, pick up the brain to be placed in position 1. Orient the brain with the side to be cut first facing upwards. Lower the brain into the OCT in the appropriate location, using the tweezers to adjust its position until it stands independently.</li>
	<li>Repeat steps 2.3&#8211;2.5 for the remaining brains/hemispheres to be frozen in positions 3&#8211;10. Avoid positioning brains/hemispheres in a symmetrical layout.</li>
	<li>Review all 9 brains in the Megabrain mold. Once the brains are standing independently, upright, and correctly oriented in the OCT (as shown in Figure 2B), add more OCT to the mold (until the top of the brains are covered). Again, ensure that there are no visible air bubbles in the OCT.</li>
	<li>Use the forceps to hold the corner of the mold, ensuring the forceps do not become partially submerged in the OCT (Figure 3A). Being careful to keep the mold level, lower the mold into the isopentane (-45&#160;&#176;C to -50&#176;C) so that the bottom third of the mold is submerged. Hold it here to allow any bubbles in the OCT to rise to the surface and away from the tissue (Figure 3B).</li>
	<li>After 30 s, lower the entire mold into the isopentane and release from the forceps, leaving the Megabrain submerged for at least 3 min (Figure 3C). Ensure that mold is kept level, so brains remain upright and equidistant (Figure 3D).</li>
	<li>After 3 min, use the forceps to remove the mold containing the frozen, OCT embedded brains (Figure 3E) from the isopentane.</li>
	<li>Immediately, wrap the mold and its contents in aluminum foil and label it with the animal numbers or Megabrain identification. Touch the frozen Megabrain as sparingly as possible to avoid thawing the tissue.</li>
	<li>This Megabrain can now be transferred to a -20&#160;&#176;C freezer and stored for a minimum of 24 h. 
	NOTE: Protocol can be paused here and the Megabrain can be stored frozen until cryosectioning takes place.</li>
</ol>
3. Preparing the Megabrain for Sectioning on the Cryostat
<ol>
	<li>Set the cabinet temperature of the cryostat to -19&#160;&#176;C. Make sure this temperature is reached before continuing. Throughout sectioning, ensure that the cabinet temperature remains between -18&#160;&#176;C and -20&#160;&#176;C.</li>
	<li>Take the Megabrain from the -20&#160;&#176;C freezer and place it into the cryostat. Also, place a chuck of the appropriate size dimensions, and two thin tipped paintbrushes into the cryostat. Leave both the Megabrain and the chuck in the cryostat for at least 15 min to come to the temperature of the cryostat.</li>
	<li>Appropriately label slides with the Megabrain identification number and the slide number. Lay these slides on a slide warmer (35&#8211;45 &#176;C).</li>
	<li>Unwrap the Megabrain from the aluminum foil. Use a razor blade to vertically cut the corners of the mold to allow easy removal of the block of OCT from the mold (Figure 4A).</li>
	<li>Dispense a thin layer (approximately 3 mm thick) of OCT onto the chuck.</li>
	<li>Quickly, with minimal contact between fingers and the OCT, place the Megabrain on the chuck, in the orientation desired. Large forceps can also be used for this step to minimize thawing. Before starting to section, leave the Megabrain mounted chuck in the cryostat for 15&#8211;20 min to allow the OCT to freeze completely.</li>
	<li>Once the base layer of OCT has frozen, apply a 2 mm thick layer OCT around the sides of the Megabrain and allow this to run down from the sides onto the chuck. This helps secure the Megabrain to the chuck. Again, before continuing, leave the Megabrain mounted chuck in the cryostat for 15&#8211;20 min to allow the OCT to freeze.</li>
	<li>Use a razor blade to remove any excess OCT from the sides or bottom of the chuck (Figure 4B).</li>
</ol>
4. Cryosectioning the Megabrain
<ol>
	<li>Before starting, ensure that a beaker containing at least 20 mL 1x PBS is accessible.</li>
	<li>Position the chuck, mounted with the Megabrain, into the chuck head on the cryostat (Figure 4B).</li>
	<li>Set the cryostat to cut at the desired thickness. The current methodology has been optimized for 10 &#181;m to 50 &#181;m sections.</li>
	<li>Trim the OCT and tissue as needed to reach the desired brain region for tissue collection (Figure 4C).</li>
	<li>When ready to collect tissue, 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 sectioned Megabrain is not attached to the anti-roll plate and does not fall off the stage Figure 4D).</li>
	<li>Gently use the paintbrushes to unroll the Megabrain section until it is lying flat on the cryostat stage (Figure 5A). (If needed, hold the OCT flat using the paintbrushes to prevent rolling).</li>
	<li>Remove slide from slide warmer and hover the slide, label-side down, over the Megabrain section on the stage allowing the section to stick to the slide.</li>
	<li>Quickly apply a drop of 1x PBS to each tissue section on the slide and do not allow these to dry out until they have been brushed flat (see step 4.9).</li>
	<li>Use a fine tipped paintbrush dipped in 1x PBS to brush out any bubbles in the tissue and unfold the tissue so it is lying flat on the slide (Figure 5B). Take care not to change the position of the brain sections and thus lose the relative position to the other tissue sections. Manipulate the tissue with care to avoid tears. Keep brain sections away from the edges of the slide, as peripheral space is required for a coverslip and PAP pen application in some staining protocols.</li>
	<li>Place the slide back onto the slide warmer and dry for 45 min. Cover as needed to prevent dust from settling on the tissue. Once dry, store the slides in at -80&#160;&#176;C until further use.</li>
</ol>

<ol>
	<li>Lyck, L., Dalmau, I., Chemnitz, J., Finsen, B., Schroder, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+markers+for+quantitative+studies+of+neurons+and+glia+in+human+neocortex.">Immunohistochemical markers for quantitative studies of neurons and glia in human neocortex.</a> Journal of Histochemistry and Cytochemistry. 56 (3), 201-221 (2008).</li><li>Fritz, P., Wu, X., Tuczek, H., Multhaupt, H., Schwarzmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+in+immunohistochemistry.+A+research+method+or+a+diagnostic+tool+in+surgical+pathology?.">Quantitation in immunohistochemistry. A research method or a diagnostic tool in surgical pathology?.</a> Pathologica. 87 (3), 300-309 (1995).</li><li>Walker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+immunohistochemistry--issues+concerning+methods,+utility+and+semiquantitative+assessment+I.">Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment I.</a> Histopathology. 49 (4), 406-410 (2006).</li><li>Evilsizor, M. N., Ray-Jones, H. F., Lifshitz, J., Ziebell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+for+immunohistochemistry+on+cryosectioned+rat+brain+tissue:+example+staining+for+microglia+and+neurons.">Primer for immunohistochemistry on cryosectioned rat brain tissue: example staining for microglia and neurons.</a> Journal of Visualized Experiments. (99), (2015).</li><li>Harrison, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolvins+AT-D1+and+E1+differentially+impact+functional+outcome,+post-traumatic+sleep,+and+microglial+activation+following+diffuse+brain+injury+in+the+mouse.">Resolvins AT-D1 and E1 differentially impact functional outcome, post-traumatic sleep, and microglial activation following diffuse brain injury in the mouse.</a> Brain, Behavior, and Immunity. 47, 131-140 (2015).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), (2012).</li><li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+the+ARRIVE+guidelines+for+reporting+animal+research.">Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.</a> PLoS Biology. 8 (6), e1000412 (2010).</li><li>Kennedy, H. S., Puth, F., Van Hoy, M., Le Pichon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+removing+the+brain+and+spinal+cord+as+one+unit+from+adult+mice+and+rats.">A method for removing the brain and spinal cord as one unit from adult mice and rats.</a> Lab Animal (NY). 40 (2), 53-57 (2011).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin+staining+of+tissue+and+cell+sections.">Hematoxylin and eosin staining of tissue and cell sections.</a> CSH Protocols. 2008, (2008).</li><li>Ji, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Impact+of+Repeated+Freeze-Thaw+Cycles+on+the+Quality+of+Biomolecules+in+Four+Different+Tissues.">The Impact of Repeated Freeze-Thaw Cycles on the Quality of Biomolecules in Four Different Tissues.</a> Biopreservation and Biobanking. 15 (5), 475-483 (2017).</li><li>Pegg, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+and+principles+of+cryopreservation.">The history and principles of cryopreservation.</a> Seminars in Reproductive Medicine. 20 (1), 5-13 (2002).</li></ol>

The authors have no disclosures.

It should be considered during this procedure that the temperature of the Megabrain and its surroundings must be constantly monitored to prevent thawing and re-freezing of the tissue. The brain can only be removed from the -20 &#176;C freezer up to 3 times as every time it is touched and left in the cryostat, with a warmer fluctuating temperature, the tissue thaws and refreezes, causing a jelly like texture and abnormal tissue integrity10. Therefore, it is optimal to cut the Megabrain all at once.
Tissue fixation and cryoprotection are key parts of this protocol to minimize ice crystal formation, reduce external microbial growth, and stop enzymatic reactions, thereby preserving tissue integrity10. If the brains are not cryoprotected with sucrose, then water within the tissue can form ice crystals during the freezing process, which shred the tissue and cause holes to form. While we suggest serial dilutions of sucrose in TBS, brains can be placed directly in 30% sucrose for an extended period (48-72 hours) to achieve similar cryoprotection. However, it is highly recommended that the sucrose solutions are made with TBS. Variations such as the use of PBS or water will result in poor cryoprotection of the Megabrain and poor tissue quality. Haematoxylin and Eosin (H&#38;E) stain can be used to assess quality of the cryoprotection and consequent freeze artifact11. Good tissue quality (with minimal holes) is essential in histological studies as the holes can disrupt the binding of antibodies as well as reducing the tissue integrity of cells with fine processes, which can affect the analysis of some stains. Avoid symmetrical placement of brains, as this can cause confusion when trying to identify the animal from which each brain was obtained.
The current technique has been optimized for coronal sections; however, it can easily be adapted to cut sagittal and transverse sections. These cutting modifications will require appropriate variation of brain placement in steps 2.5 through 2.7. It should be noted that these adaptions may reduce the number of brains that can fit into one embedding mold.
A key modification of this technique is the blank space at position number 1 (as shown in Figure 1). This space is designed to allow easy identification of the Megabrain orientation, and therefore identify the animal associated with each brain.
This technique has been modified to allow tissue from a larger number of animals to be frozen and cut simultaneously in the Megabrain. The size of the rodent brain is dependent on sex, age and species therefore; the number of brains that fit in the mold can vary. It is not advised to segregate different ages, sexes, or treatment groups into separate Megabrains. Although this can ensure size uniformity, it may also increase bias and subjectivity during imaging and analysis. In addition, there is inevitable variance between slides during immunohistochemistry in any protocol, which could lead to staining inconsistency between age/sex groups.
A rectangular, larger chuck was used, in place of a circular one, which provided increased support behind the Megabrain.
As the mold and its contents were of a larger volume than that of a singular brain in an OCT mold, more time was required to freeze the brain in the isopentane. Through trial and error, a length of time that provided good freeze quality (3.5 minutes) was determined. The Megabrain can be left in the isopentane for longer; however, the temperature must stay in the range of -45 &#176;C to -50&#176;C to avoid cracking the OCT.
As there are many separate pieces of tissue on the Megabrain slide, it is possible that tissue can dry out and consequently become more brittle during the brushing steps. To combat this issue, a drop of PBS should be put on each individual brain section to keep it moist until it is brushed out.
The technique of hemisecting the brain, for a study in which this is required, is critical to obtaining good results. When hemisecting the brain, it is important that this bisection is done precisely to ensure all brain regions are intact with the correct hemisphere. If the cerebellum or olfactory bulbs are not relevant to the study, they can be removed. Removing this &#39;excess&#39; tissue reduces the amount of tissue that will need to be trimmed when cryosectioning. Removing the cerebellum allows the brains to stand up more easily in the OCT before freezing.
While being brushed out, keep the tissue sections away from the edge of the slide. This allows room for a line of PAP pen, a hydrophobic barrier, to surround the tissue, which is a requirement of some immunohistochemistry protocols. Additionally, space is needed for the coverslip.
If the hemisphere/brain falls or tilts during the Megabrain freezing, then the sections will not be co-planer. A similar outcome can result if one brain is larger than the others in a single Megabrain. To overcome this, more slides can be selected to stain where the brain(s) in question are at the appropriate area of interest.
One limitation of this technique is that as all of the tissue is frozen in one block of OCT; if there is technical error, such as a bad freeze due to an incorrect temperature, then the tissue from multiple animals will be affected (5-9 animal brains instead of just 1). This is more of a problem when using the full brain and not hemisected sections, because the remaining hemisphere can be frozen and sectioned singly to obtain the desired sections.
Another limitation to this method is that when adjusting the cryostat to cut the Megabrain, a best-fit approach must be taken. This is due to all brains being at slightly different angles to one another, whereas this is not an issue when sectioning a singularly frozen brain. The effects of this can be minimized during freezing by ensuring brains are upright and of a similar size.
One of the biggest benefits of using this technique over freezing brains singularly is that it shortens the overall time spent freezing and cutting brains by up to 90%, if 9 are being cut at the same pace as 1 brain. The other key advantage of this technique is that it allows a smaller number of slides that contain tissue from the required animals to be stained. This increases the reliability and uniformity of immunohistochemical staining, since all slides are stained in the same round. There can be inconsistency between slides stained at the same time, but a Megabrain can allow several study brains to be represented on a single slide, thus reducing variability and increasing the validity of the analysis. Having consistent staining is a key advantage in any histological study and a major significance of using this technique. Lastly, fewer slides also incur lower supply costs.
This technique could be easily modified for cutting sagittal sections of a rodent brain. It could also be adapted for use in cryosectioning the tissue from different animal models and different types of tissue, such as muscle or liver samples. Adaptions to the protocol would have to be made, including optimization of the amount of solutions used, size of the cryostat blade/stage, and the volume of the embedding mold.
This technique could also be adapted for free-floating techniques, where the protocol would remain the same until step 4.6. At this step, brains would need to be separated and lifted into separate plate wells to be stained. Extra care would need to be taken not to confuse the brain slices in the sorting process.

Here, we present the protocol for a novel method, which we call Megabrain, developed to cryosection multiple rodent brains simultaneously for downstream immunohistochemical procedures. A Megabrain allows for the production of single slides containing tissue from multiple animals. This technique has been optimized to cut coronal sections from 9 adult rat hemispheres, or 5 adult full brains, simultaneously. Therefore, the technique is most applicable in large immunohistochemical studies or other analyses done on slide-mounted brain tissue from a large cohort of animals.
Immunohistochemistry involves the use of specific antibodies directed against proteins of interest to understand and characterize their expression and cellular changes in specific tissue1,2,3. The use of immunohistochemistry is prevalent in neuroscience research, among other scientific disciplines, aiding in the cellular and molecular understanding of the brain4. Large-scale studies involving many animals and brain sections can be both resource and time intensive. As such, there is a multifaceted rationale behind the development of the Megabrain: to reduce time spent cryosectioning, mounting, and staining tissue, while consequently using less reagents. Moreover, the ability to streamline the process and stain multiple brains in the same round helps to alleviate some of the variability between staining batches, a limitation of immunohistochemistry3. In addition, sectioning a Megabrain is a time saving alternative to sectioning individual frozen rodent brains and allows for rapid comparison of tissue between animals or even treatment groups by microscopy techniques.

<table><tbody><tr><td>Andwin scientific tissue-tek CRYO-OCT compound (case of 12)</td><td>Fisher Scientific</td><td>14-373-65</td><td></td></tr><tr><td>Thermo Scientific Shandon Peel-A-Way Disposable Embedding Molds</td><td>Fisher Scientific</td><td>18-41</td><td></td></tr><tr><td>Fisherbrand High Precision Straight Broad Strong Point Tweezers/Forceps</td><td>Fisher Scientific</td><td>12-000-128</td><td></td></tr><tr><td>Fisherbrand 20 mL HDPE Scintillation Vials with Polypropylene Cap</td><td>Fisher Scientific</td><td>03-337-23</td><td></td></tr><tr><td>Sucrose, poly bottle 2.5 kg</td><td>Fisher Scientific</td><td>S2-212</td><td>Both 15% and 30% sucrose concentrations need to be made up.</td></tr><tr><td>2-Methylbutane (Certified), Fisher Chemical</td><td>Fisher Scientific</td><td>O3551-4</td><td></td></tr><tr><td>PYREX Tall-Form Beakers</td><td>Fisher Scientific</td><td>02-546E</td><td></td></tr><tr><td>Fisherbrand General Purpose Liquid-in-Glass Partial Immersion Thermometers (-50 to +50 &amp;deg;C)</td><td>Fisher Scientific</td><td>13-201-642</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Scoopula Spatula</td><td>Fisher Scientific</td><td>14-357Q</td><td></td></tr><tr><td>STANLEY Razor Blade</td><td>Grainger</td><td>4A807</td><td></td></tr><tr><td>Edge-Rite Microtome blades</td><td>Fisher Scientific</td><td>14-070-60</td><td></td></tr><tr><td>Microscope slides (1&quot; frost) - white</td><td>Fisher Scientific</td><td>22-034-979</td><td></td></tr><tr><td>Gibco&amp;nbsp;PBS (Phosphate Buffered Saline) 10x, pH 7.2</td><td>Fisher Scientific</td><td>70-013-032</td><td>Dilute to 1X&amp;nbsp; before use</td></tr><tr><td>15 piece fine paint brushes</td><td>Amazon</td><td>B079J12ZRV</td><td></td></tr><tr><td>PAP pen</td><td>abcam</td><td>ab2601</td><td></td></tr><tr><td>Chuck Shown in Figure 4 was custom made by a lab technician, however similar sizes are available to order from other companies commercially.</td><td>Electron Microscopy Sciences</td><td>EMS065</td><td></td></tr></tbody></table>

simultaneous cryosectioning of multiple rodent brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

A positive end result to this procedure is tissue that lies flat on the slide, with no bubbles or tears, in the orientation in which they were frozen. Tissue sections are evenly spaced apart and easily identifiable due to good placement of the brain in the OCT and good notation as demonstrated in Figure 1. Assuming that the brain tissue was collected from animals of a similar age, and that the tissue was properly aligned in the OCT, sections collected on a slide should represent a similar coronal plane, allowing comparison of brain regions between animals. Provided that the tissue has frozen well, with minimal freeze artifact, H&#38;E stain can be carried out to assess the cryoprotection quality9. Immunohistochemistry can be carried out as demonstrated with the staining shown in Figure 6.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58513/58513fig1.jpg" /> 
Figure 1: Representative diagram of a suggested Megabrain layout. This shows the position notation of tissue derived from 9 different animals. The number system is designed as demonstrated; however, any numbering system may be used. &#39;X&#39; is representative of the blank space designed to clearly show the orientation in which the tissue was frozen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58513/58513fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58513/58513fig2.jpg" /> 
Figure 2: Incorrectly and correctly positioned brains in mold. (A) Brain hemispheres poorly aligned in OCT from the top and side. (B) Well-aligned brain hemispheres in OCT from the top and side. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58513/58513fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58513/58513fig3.jpg" /> 
Figure 3: Stages of brain freezing.&#160;(A) Megabrain before being submerged in -45 &#176;C isopentane. (B) Megabrain when submerged in isopentane. (C) Megabrain fully submerged in isopentane. (D) Megabrain being lowered into isopentane, demonstrating that brains should stay upright and equidistant from one another during this process. (E) A frozen Megabrain in an embedding mold. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58513/58513fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58513/58513fig4.jpg" /> 
Figure 4: Key stages in cutting a Megabrain.&#160;(A) Megabrain being removed from embedding mold in cryostat. (B) Chuck mounted with Megabrain. (C) Trimmed Megabrain. (D) Megabrain section on cryostat stage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58513/58513fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58513/58513fig5.jpg" /> 
Figure 5: Pre- and post-mounted Megabrain section.&#160;(A) Frozen Megabrain section (40 &#181;m) on cryostat stage. (B) Glass slide mounted with Megabrain tissue. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58513/58513fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58513/58513fig6.jpg" /> 
Figure 6: Immunohistochemistry of a Megabrain slide stained with Iba1. (A) Tissue taken from a Megbrain shows uniform staining between different brains in a study.&#160;(B) 20x microscopic images taken from the somatosensory barrel field (S1BF) cortex region of each mounted brain, showing consistent ionized calcium-binding adapter molecule 1 (Iba1) staining of microglia between brains. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58513/58513fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Simultaneous Cryosectioning of Multiple Rodent Brains at JoVE.com

Simultaneous Cryosectioning of Multiple Rodent Brains

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

simultaneous-cryosectioning-of-multiple-rodent-brains

Nanyang Technological University

Research

JoVE Journal

Neuroscience

23.4K Views.  University of Arizona College of Medicine. Deze tijdbesparende methode vermindert de variabiliteit tussen rondes van immunohistochemische procedures in neurowetenschappelijke studies om de visualisatie van anatomie en lokale eiwitten en weefsel te optimaliseren. Het belangrijkste voordeel van deze techniek is dat het vermindert de tijd die nodig is om cryosection en monteren hersenweefsel door het combineren van meerdere hersenen in een enkele bevroren blok. Terwijl deze techniek is geoptimaliseerd voor coronale secties van knaagdier ...

Video: Simultaneous Cryosectioning of Multiple Rodent Brains

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection

The present protocol describes a standardized paradigm for rodent brain tumor resection and tissue preservation. In clinical practice, maximal tumor resection is the standard-of-care treatment for most brain tumors. However, most currently available preclinical brain tumor models either do not include resection, or utilize surgical resection models that are time-consuming and lead to significant postoperative morbidity, mortality, or experimental variability. In addition, performing resection in rodents can be daunting for several reasons, including a lack of clinically comparable surgical tools or protocols and the absence of an established platform for standardized tissue collection. This protocol highlights the use of a multi-functional, non-ablative resection device and an integrated tissue preservation system adapted from the clinical version of the device. The device applied in the present study combines tunable suction and a cylindrical blade at the aperture to precisely probe, cut, and suction tissue. The minimally invasive resection device performs its functions via the same burr hole used for the initial tumor implantation. This approach minimizes alterations to regional anatomy during biopsy or resection surgeries and reduces the risk of significant blood loss. These factors significantly reduced the operative time (&#60;2 min/animal), improved postoperative animal survival, lower variability in experimental groups, and result in high viability of resected tissues and cells for future analyses. This process is facilitated by a blade speed of ~1,400 cycles/min, which allows the harvesting of tissues into a sterile closed system that can be filled with a physiologic solution of choice. Given the emerging importance of studying and accurately modeling the impact of surgery, preservation and rigorous comparative analysis of regionalized tumor resection specimens, and intra-cavity-delivered therapeutics, this unique protocol will expand opportunities to explore unanswered questions about perioperative management and therapeutic discovery for brain tumor patients.

The present protocol describes a standardized resection of brain tumors in rodents through a minimally invasive approach with an integrated tissue preservation system. This technique has implications for accurately mirroring the standard of care in rodent and other animal models.

The present protocol describes a standardized paradigm for rodent brain tumor resection and tissue preservation. In clinical practice, maximal tumor ...

Cancer Research

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Biology

Optimizing Stereotaxic Microinjection Accuracy in Mice

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their choice of stereotaxic injection parameters on mouse brain atlases or published materials that employed various populations/ages of mice and different stereotaxic equipment, necessitating further validation of the stereotaxic coordinate parameters. The efficacy of calcium imaging, chemogenetic, and optogenetic manipulations relies on the precise expression of reporter genes within the region of interest, often requiring several weeks of effort. Thus, it is a time-consuming task if the coordinates of the target brain region are not verified in advance. Using an appropriate dye instead of a virus and implementing cryosectioning, researchers can observe the injection site immediately following dye administration. This facilitates timely adjustments to coordinate parameters in cases where discrepancies exist between the actual injection site and the theoretical position. Such adjustments significantly enhance the accuracy of viral expression within the target region in subsequent experiments.

Author Spotlight: Accelerating Cognitive Impairment Research in Mice Through Stereotaxic Injection and Cost-Effective Dyes

The present protocol describes a practical strategy to expedite the verification step of stereotaxic injection coordinates before conducting viral tracing using dyes and frozen sections.

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their ...

Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue

Source: Comba, A. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60939/laser-capture-microdissection-glioma-subregions-for-spatial-molecular">Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the sectioning of a frozen mouse brain tumor tissue using a cryostat. The frozen brain tissue sections so obtained are stored at low temperatures until further analysis.

Source: Comba, A. et al. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, ...

Simultaneous Cryosectioning of Multiple Rodent Brains

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