We thank Wei Liu for his technical assistance. This work is funded by ANPCyT PICT 2017-2497 and PICT 2018-4113.

All experimental procedures followed the guidelines of the Canadian Council on Animal Care.
1. Sample collection and preparation
<ol>
	<li>Prepare 500 mL of 4% paraformaldehyde (PFA).
	<ol>
		<li>Under an extraction flux in a cabinet, add 20 g of PFA powder to 250 mL of 1x phosphate-buffered saline (PBS) in a 1 L glass beaker. Place the beaker with a magnet on a magnetic stirring plate.</li>
		<li>Stir while heating. With a thermometer, constantly check the temperature of the solution to keep it under 60 &#176;C.</li>
		<li>With a plastic Pasteur pipette, add drops of 1 M NaOH to the solution to dissolve the PFA. Once the PFA powder has completely dissolved, cool the solution at room temperature (RT).</li>
		<li>Adjust the volume to 500 mL with 1x PBS. Adjust the pH to 7.2-7.3 by adding drops of 1 M HCl with a plastic Pasteur pipette.</li>
		<li>Using a paper filter and a glass funnel, filter the solution in a 1 L glass bottle.</li>
		<li>Close the glass bottle, remove it from the flux cabinet, and store in a refrigerator at -4 &#176;C.</li>
	</ol>
	</li>
	<li>Collect brain samples.
	<ol>
		<li>To obtain samples, sacrifice mouse neonates the morning following their birth, at 0.5 days of age (P 0.5), using surgical scissors to decapitate them.</li>
		<li>Place the scissors in the neck of the neonate while holding the body, and perform a single and clean cut.</li>
	</ol>
	</li>
	<li>Fixate the samples in PFA.
	<ol>
		<li>Fill a 50 mL conical tube with 40 mL of 4% PFA.</li>
		<li>Immerse each head in the tube containing PFA as a fixation agent. For this action, use a Paton spatula or a spoon.</li>
		<li>Store the conical tubes in the refrigerator at -4 &#176;C for 48 h, then change to 40 mL of 1x PBS with 0.1% sodium azide as a preservative. Maintain refrigerated at -4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Sample staining
<ol>
	<li>Prepare Lugol&#39;s solution (3.75% w/v).
	<ol>
		<li>Under an extraction flux in a cabinet, in a 1 L glass beaker, dissolve 10 g of Kl and 5 g of I2 in 400 mL of distilled water, with constant mixing using a magnetic stirrer.</li>
	</ol>
	</li>
	<li>Immerse each head in a 50 mL conical tube containing 40 mL of Lugol&#39;s solution.
	<ol>
		<li>For neonates of an approximate body size of 1.3 g, leave the samples immersed for 15-20 h with constant mixing using an orbital shaker.</li>
		<li>Place the samples in a 15 mL conical tube containing 10 mL of 1% agarose for 30 min before scanning.</li>
	</ol>
	</li>
</ol>
3. Micro-CT scanning
NOTE: Micro-CT scanning requires specific equipment. There are different options for this kind of scanner, and details on acquisition depend on the characteristics of the used equipment. Dr. Hallgr&#237;msson&#39;s lab counts on some alternatives of micro-CT scanners. Here, the protocol is based on the use of a basic desktop scanner, which is among the more accessible small animal imaging machines used by labs around the world. If the scanning objective is the skull, skip the steps in section 2 of the protocol and proceed to section 3. After scanning the skull, the staining process and the scanning of the brain could be performed to obtain images from the same specimens of both the brain and the skull.
<ol>
	<li>Place the sample in the micro-CT holder.
	<ol>
		<li>Place the sample in a 50 mL conical tube. Fix the sample to avoid movements during the scanner session. For this purpose, some polyurethane material can be used to fill the empty parts of the tube.</li>
		<li>Open the equipment and place the tube with the sample in the micro-CT holder.</li>
		<li>Log into the micro-CT control panel and register the sample to be scanned. This will automatically generate a sample number. In addition to the number, fill out the &#34;Name&#34; field. Both the sample name and number should be recorded elsewhere (e.g., in a spreadsheet) in case the raw data needs to be retrieved again.</li>
		<li>Start the scanning program. Enter the sample name or number and select a Control file, which should contain the scanning parameters.</li>
	</ol>
	</li>
	<li>Scan the sample.
	<ol>
		<li>In the screen of the micro-CT software, set the following parameters: isotropic voxel size of 0.012 mm, 45 kVp, 177 lA, 800,000 ms integration time, and 500 projections per 180&#176;.</li>
		<li>Set the scanning region by selecting Scout View. Once the reference image appears, press Reference Line to set the region. Move the solid green line to the edge of the sample, then hold Shift while dragging the cursor to extend the scan region to the other edge of the sample.</li>
		<li>Select OK to begin the scan.</li>
	</ol>
	</li>
	<li>Evaluate and export the scan.
	<ol>
		<li>Open up the micro-CT scanner Evaluation Program and click on Select Sample and Measurement. In the Filter field, type the sample name, select the sample, and select the measurement file.</li>
		<li>The file will load up as slices, the number of which depends on the initial scanning window. Click on the Load All button, which will load every slice and facilitate movement between slices later on.</li>
		<li>Select Tasks &#62; Evaluation 3D to initialize a cropping box around the slices. A 3D-Evaluation prompt will appear with a number of fields, including Task/Evaluation, VOI (Volume of Interest), Start X, Y, Z, and Dimension X, Y, Z.</li>
		<li>Set the Task &#62; Evaluation 3D field to the appropriate script, as a variety of evaluation tasks can be performed, such as reconstruction, segmentation, morphometry (e.g., bone mineral density), and file conversions or transfers. The Default Evaluation script can be used as a guide.</li>
		<li>After selecting the script, adjust the VOI box on the main image. Start on the first slice. Ensure that the box contains the anatomy to be evaluated. Move the VOI by right-clicking on one of the small white squares toward the box edge and resizing the VOI using the middle mouse button. Alternatively, adjust the Dimension fields numerically.</li>
		<li>Pass through the sample using the scroll bar underneath the image to ensure that all of the anatomies have been captured in the VOI box. Click on XY, XZ, or YZ near the top left of the software to change the orientation.</li>
		<li>Select Start Evaluation. Depending on the Task Evaluation script, this will generate a 3D .aim image file with a corresponding .txt header file. Additionally, a path can be specified in the script to File Transfer Protocol (FTP) the image to a particular location.</li>
	</ol>
	</li>
</ol>
4. Image processing
<ol>
	<li>Open the images in the image processing software. 
	NOTE: Image processing can be carried out using various software. This protocol includes the basic steps in a commercial software, which is widely accepted in the area and has versatile tools for basic and advanced processing. In addition, there are different acceptable formats for reconstructed micro-CT images. This aspect usually depends on the equipment used for acquisition and its software. Here, image processing is carried out with .bmp images. The same procedure can be applied for .tiff images and other types. Since the acquisition is carried out in a scanner with .aim as the default format, a conversion from .aim to .bmp is first presented.
	<ol>
		<li>To open .aim files, select File &#62; Open Data and choose the file. After that, a window with the title File Format Selection will open. Select Raw Data.</li>
		<li>A window with the title Raw Data Parameters will open. On this window, fill in the parameters following the information on the header .txt file that is generated for each .aim file. For Data Type, choose 16-bit for header complete with the number after Image data starts at byte offset. Also, set the dimensions and voxel size using the information on the header .txt file. Once the parameters are complete, press OK.</li>
		<li>If conversion from .aim to .bmp is needed, in Main Panel &#62; Project View, right-click on the .aim object and select the option Convert &#62; Convert Image Type. On Properties, select 8-bit as the Output Type. Select Apply.</li>
		<li>Right-click on the new 8-bit object. Select Save as. Choose .bmp and save.</li>
		<li>To open the .bmp file, select File &#62; Open Data and choose the file. After that, a window with the parameters of the image opens. Confirm the parameters and accept if correct.</li>
		<li>In Main Panel &#62; Project View, a new object is created with the name of the .bmp files. Right-click on this object and select the option Ortho Slice. In Properties, select the slice number and the orientation plane to see.</li>
	</ol>
	</li>
	<li>Obtain 3D volumes of the complete head.
	<ol>
		<li>In Main Panel &#62; Project View, right-click on the image object. Select Interactive Thresholding option &#62; Create. In Properties, change the values for Minimum RGB and Maximum RGB until the part of the image corresponding to the head of the animal is selected. Select Apply.</li>
		<li>In Main Panel &#62; Project View, a thresholded object is created. Right-click on this object and, in Properties, select Isosurface. In Properties, choose a threshold to visualize the surface and click Apply. This threshold needs to be chosen empirically, then different values might need to be examined.</li>
	</ol>
	</li>
	<li>Digitizing point coordinates
	<ol>
		<li>3D point coordinates can be digitized both based on slices or extracted surfaces. For the first option, right-click in the image option and select Slice. In Translate, choose the desired slice in the axial plane. If the plane is not correctly placed, click Options &#62; Rotate in Properties to establish the right orientation.</li>
		<li>To digitize landmarks, right-click in Main Panel &#62; Project View and select Create Object &#62; Points and Lines &#62; Landmarks. Right-click the new Object Landmarks and select Landmark View. In Landmark View, modify the size of the point in Size. To add landmarks in Properties, use the option Add in Edit Mode. 
		NOTE: For neonates, a set of landmarks and semilandmarks around the axial and sagittal curves of the brain was previously published14.</li>
		<li>First, choose the axial plane that crosses the most rostral point of the olfactory bulbs and the most caudal point of the cortex.</li>
		<li>Select the Object Landmarks and, in Properties, select Landmark Editor. Using the arrow, click on the place the point is to be digitized.</li>
		<li>In the proposed set of landmarks, 24 points are digitized in the selected axial plane. Digitize the first in the most caudal point of the brain at the midline.</li>
		<li>Digitize five points equally distributed around the curve (semilandmarks) until the next landmark at the intersection between the midbrain and the cortex.</li>
		<li>Then, digitize eight points (semilandmarks) around the cortex.</li>
		<li>Digitize a new landmark at the intersection between the cortex and the olfactory bulbs. Then, digitize four points (semilandmarks) around the olfactory bulbs.</li>
		<li>Digitize a new landmark at the most rostral point of the olfactory bulbs at the midline. Then, digitize two points (semilandmarks) in between both olfactory bulbs.</li>
		<li>Digitize a new landmark at the most caudal point of the olfactory bulbs at the midline.</li>
		<li>For sagittal points, establish a slice in the right side of the brain at a parasagittal plane where the brain is most caudally prominent.</li>
		<li>In the proposed set of landmarks, 33 points are digitized in the selected parasagittal plane. Digitize the first in the intersection between the diencephalon and the hindbrain. Then, digitize nine points (semilandmarks) in the ventral limit of the brain.</li>
		<li>Digitize a new landmark at the most rostral point of the olfactory bulb. Then, digitize three points (semilandmarks) around the olfactory bulbs.</li>
		<li>Digitize a new landmark in the intersection between the olfactory bulb and the cortex. Then, digitize three points (semilandmarks) around the olfactory bulbs.</li>
		<li>Digitize a new landmark in the intersection between the olfactory bulb and the cortex. Then, digitize seven points (semilandmarks) around the cortex.</li>
		<li>Digitize a new landmark in the intersection between the cortex and the midbrain. Then, digitize seven points (semilandmarks) around the cortex.</li>
		<li>Digitize a new landmark in the intersection between the cortex and the midbrain. Then, digitize six points (semilandmarks) around the midbrain.</li>
		<li>Digitize a new landmark in the intersection between the midbrain and the cerebellum. Then, digitize two points (semilandmarks) around the cerebellum.</li>
		<li>Digitize a new landmark in the intersection between the cerebellum and the hindbrain.</li>
		<li>Save the digitized points. Right-click on the Object Landmarks and choose the option Save Data.</li>
	</ol>
	</li>
	<li>Analyze the point coordinates. 
	NOTE: Once the point coordinates are digitized for a set of specimens, basic analyses can be performed using geometric morphometrics tools to obtain size (centroid size) and shape variables (shape or Procrustes coordinates). Analyses are carried out in a free open software environment, which is especially suitable for statistical analyses.
	<ol>
		<li>Using a notepad, build a file containing the coordinates of all the digitized specimens. For this purpose, follow the TPS format, as described in&#160;https://morphometrics.uk/MorphoJ_guide/frameset.htm?index.htm.</li>
		<li>Open the statistical software. Select File &#62; Change Dir to choose the directory where the .tps file is saved.</li>
		<li>Select Packages &#62; Install packages. Choose one Cran Mirror. Look for geomorph and install it.</li>
		<li>Load packages by typing in the console: library(geomorph) and library(Morpho).</li>
		<li>Load the dataset by typing in the console: dataset &#60;- readland.tps(file=&#34;NAME_OF_FILE.tps&#34;, specID=&#34;ID&#34;).</li>
		<li>Perform generalized Procrustes analysis by typing in the console: GPA&#60;- gpagen(dataset).</li>
		<li>Obtain the centroid size by typing in the console: CS&#60;-GPA$Csize.</li>
		<li>Obtain Procrustes coordinates by typing in the console: ProcCoord&#60;- GPA$coords.</li>
		<li>Plot the Procrustes coordinates and the mean shape by typing in the console: plotAllSpecimens(ProcCoord).</li>
	</ol>
	</li>
	<li>Segmentation of ROIs 
	NOTE: To segment the brain and obtain a 3D reconstruction, manually identify the brain tissues in each slice. The same procedure can be applied to specific ROIs, such as the olfactory bulbs, cortex, etc.
	<ol>
		<li>Right-click on Main Panel &#62; Project View and select Create Object &#62; Label Field.</li>
		<li>Selecting the Label Field object, press the Segmentation Editor option in Properties.</li>
		<li>In Materials, add a new material and, if desired, rename it as Brain.</li>
		<li>In Selection, choose the option Current Slice to segment the tissue corresponding to the brain in each slice.</li>
		<li>Using the options present in Tools, select the brain tissue in each slice. For this case, the Magic Wand and the Brush are the most suitable.</li>
		<li>Once the selection is made in each slice, press Add in Selection.</li>
		<li>When selection in all slices is finished, return to Project View in Main Panel and right-click on Label Field to select Generate Surface, then click Apply.</li>
		<li>Once the surface is obtained, export it by applying Extract Surface.</li>
		<li>To obtain the volume of the segmented structures, in Main Panel &#62; Project View, select the object that contains the label, right-click, and select Measure and Analyze &#62; Volume Fraction. Then, click Apply.</li>
	</ol>
	</li>
</ol>

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

<ol>
	<li>Altman, A. R., 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=Quantification+of+skeletal+growth,+modeling,+and+remodeling+by+in+vivo+micro-computed+tomography.">Quantification of skeletal growth, modeling, and remodeling by in vivo micro-computed tomography.</a> Bone. 81, 370-379 (2015).</li><li>Wehrle, E., 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=Spatio-temporal+characterization+of+fracture+healing+patterns+and+assessment+of+biomaterials+by+time-lapsed+in+vivo+micro-computed+tomography.">Spatio-temporal characterization of fracture healing patterns and assessment of biomaterials by time-lapsed in vivo micro-computed tomography.</a> Scientific Reports. 11 (1), 8660 (2021).</li><li>Ar&#237;stide, 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=Brain+shape+convergence+in+the+adaptive+radiation+of+New+World+monkeys.">Brain shape convergence in the adaptive radiation of New World monkeys.</a> Proceedings of the National Academy of Sciences. 113 (8), 2158-2163 (2016).</li><li>Paluh, D. J., Stanley, E. L., Blackburn, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+hyperossification+expands+skull+diversity+in+frogs.">Evolution of hyperossification expands skull diversity in frogs.</a> Proceedings of the National Academy of Sciences. 117 (15), 8554-8562 (2020).</li><li>de Crespigny, A., 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=3D+micro-CT+imaging+of+the+postmortem+brain.">3D micro-CT imaging of the postmortem brain.</a> Journal of Neuroscience Methods. 171 (2), 207-213 (2008).</li><li>Gignac, P. M., 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=Diffusible+iodine-based+contrast-enhanced+computed+tomography+(diceCT):+an+emerging+tool+for+rapid,+high-resolution,+3-D+imaging+of+metazoan+soft+tissues.">Diffusible iodine-based contrast-enhanced computed tomography (diceCT): an emerging tool for rapid, high-resolution, 3-D imaging of metazoan soft tissues.</a> Journal of Anatomy. 228 (6), 889-909 (2016).</li><li>Wong, M. D., Dorr, A. E., Walls, J. R., Lerch, J. P., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+mouse+embryo+atlas+based+on+micro-CT.">A novel 3D mouse embryo atlas based on micro-CT.</a> Development. 139 (17), 3248-3256 (2012).</li><li>Gonzalez, P. N., 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=Chronic+protein+restriction+in+mice+impacts+placental+function+and+maternal+body+weight+before+fetal+growth.">Chronic protein restriction in mice impacts placental function and maternal body weight before fetal growth.</a> PLoS One. 11 (3), 0152227 (2016).</li><li>Watanabe, A., 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=Are+endocasts+good+proxies+for+brain+size+and+shape+in+archosaurs+throughout+ontogeny.">Are endocasts good proxies for brain size and shape in archosaurs throughout ontogeny.</a> Journal of Anatomy. 234 (3), 291-305 (2019).</li><li>Gignac, P. M., Kley, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+diceCT+imaging+for+high-throughput+comparative+neuroanatomical+studies.">The utility of diceCT imaging for high-throughput comparative neuroanatomical studies.</a> Brain, Behavior and Evolution. 91 (3), 180-190 (2018).</li><li>Anderson, R., Maga, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+procedure+for+rapid+imaging+of+adult+mouse+brains+with+microCT+using+iodine-based+contrast.">A novel procedure for rapid imaging of adult mouse brains with microCT using iodine-based contrast.</a> PLoS One. 10 (11), e0142974 (2015).</li><li>Gignac, P. M., O'Brien, H. D., Sanchez, J., Vazquez-Sanroman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+imaging+of+the+rat+brain+using+an+integrated+diceCT+and+histology+workflow.">Multiscale imaging of the rat brain using an integrated diceCT and histology workflow.</a> Brain Structure &amp; Function. 226 (7), 2153-2168 (2021).</li><li>Wong, M. D., Spring, S., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+stabilization+of+tissue+for+embryo+phenotyping+using+micro-CT+with+iodine+staining.">Structural stabilization of tissue for embryo phenotyping using micro-CT with iodine staining.</a> PLoS One. 8 (12), e84321 (2013).</li><li>Barbeito-Andr&#233;s, J., 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=Congenital+Zika+syndrome+is+associated+with+maternal+protein+malnutrition.">Congenital Zika syndrome is associated with maternal protein malnutrition.</a> Science Advances. 6 (2), (2020).</li><li>Handschuh, S., Gl&#246;smann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryo+phenotyping+using+X-ray+microCT.">Mouse embryo phenotyping using X-ray microCT.</a> Frontiers in Cell and Developmental Biology. 10, 949184 (2022).</li><li>Turnbull, D. H., Mori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+in+mouse+developmental+biology.">MRI in mouse developmental biology.</a> NMR in Biomedicine. 20 (3), 265-274 (2007).</li><li>Qiu, L. R., 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=Mouse+MRI+shows+brain+areas+relatively+larger+in+males+emerge+before+those+larger+in+females.">Mouse MRI shows brain areas relatively larger in males emerge before those larger in females.</a> Nature Communications. 9, 2615 (2018).</li><li>Lerch, J. P., Sled, J. G., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+phenotyping+of+genetically+altered+mice.">MRI phenotyping of genetically altered mice.</a> Methods in Molecular Biology. 711, 349-361 (2011).</li><li>Gonzalez, P. N., Kristensen, E., Morck, D. W., Boyd, S., Hallgr&#237;msson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+growth+hormone+on+the+ontogenetic+allometry+of+craniofacial+bones.">Effects of growth hormone on the ontogenetic allometry of craniofacial bones.</a> Evolution &amp; Development. 15 (2), 133-145 (2016).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+developmental+biology:+a+versatile+tool+for+high-contrast+3D+imaging+at+histological+resolutions.">MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions.</a> Developmental Dynamics. 238 (3), 632-640 (2009).</li><li>Vickerton, P., Jarvis, J., Jeffery, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration-dependent+specimen+shrinkage+in+iodine-enhanced+microCT.">Concentration-dependent specimen shrinkage in iodine-enhanced microCT.</a> Journal of Anatomy. 223 (2), 185-193 (2013).</li><li>Dawood, Y., 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=Reducing+soft-tissue+shrinkage+artefacts+caused+by+staining+with+Lugol's+solution.">Reducing soft-tissue shrinkage artefacts caused by staining with Lugol's solution.</a> Scientific Reports. 11, 19781 (2021).</li></ol>

The authors declare that they have no competing interests.

In this work, a concise protocol to scan neonatal brain tissues of mice using micro-CT with a contrast agent is introduced. In addition, it includes simple procedures to obtain quantitative and qualitative outputs. Building on these methods, further alternative or complementary analyses can be performed.
As shown in the protocol, micro-CT images can be analyzed in different ways. In previous studies, our group estimated the size and shape variation in perinatal brains of mice by digitizing coo...

Micro-computed tomography (micro-CT) imaging is a valuable tool for different fields of research. In biology, it is especially suitable for bone research because of X-ray absorption in mineralized tissues. Due to this feature, diverse questions regarding bone development1, metabolism2, and evolution3,4, among other topics, have been approached with the assistance of micro-CT. In 2008, de Crespigny et al.5 showed that micro-CT images of adult mouse and rabbit brains could be obtained using iodine as a contrast agent. This work opened a new application for this imaging technique, since iodine allowed the acquisition of images from soft tissues which otherwise would be insensitive to X-rays. Thus, the general goal of combining micro-CT and an iodine-based contrast agent is to obtain high-resolution images, in which soft tissues can be distinguished and identified at a meso or macro anatomical level.
This technique has notable potential for studies that require detailed ex vivo phenotypic characterization of small specimens, such as mouse embryos, which are widely used in experimental designs6. Iodine contrast in combination with micro-CT imaging has been used to obtain volumetric quantifications of organs7 and landmark three-dimensional (3D) structures8,9. In recent years, micro-CT scanning of stained samples has been applied to describe brain phenotypic features of rodents10, and different improvements to the technique have been proposed. For adult brains, a protocol of 48 h of immersion in iodine, with a previous step of perfusion with a hydrogel, was found to produce images of high quality11. Gignac et al.12 expanded the limits of this technique by showing that rat brains stained with iodine could be processed to perform routine histological techniques. Similarly, these procedures demonstrate promising results for embryonic and pre-weaning rodent brains8,13,14,15.
Although neuroscience has largely applied microscope-based techniques to assess different structural and functional aspects of brain development, such studies are more suitable for characterizing specific cell populations or spatially limited structures. Conversely, micro-CT imaging allows the description of whole structures and the acquisition of 3D models that preserve relevant spatial information, which is complementary to microscopic techniques. Magnetic resonance imaging (MRI) is also a standard technique applied to explore the structural features of small animals16,17,18. However, micro-CT, with the use of a contrast agent, has two main advantages for ex vivo fixed samples: micro-CT scanners are largely less expensive and easy to operate, and allow a higher spatial resolution than MRI12.
This work aims to describe the procedure to obtain high-resolution images from neonatal mouse brains using micro-CT scanning after staining with Lugol&#39;s solution, an iodine-based contrast agent. A comprehensive protocol is presented, which starts with preliminary stages such as sample collection and fixation of tissues, and goes through staining, micro-CT image acquisition, and standard processing. Image processing includes the segmentation of a 3D volume of the complete head, as well as of the brain, and the selection of specific anatomical planes to digitize point coordinates that could be then used in morphometric analyses. Although the focus here is the neonatal mouse brain, similar strategies can be applied to other soft tissues. Thus, the protocol presented here is flexible enough to be applied, with subtle modifications, to other types of samples.

<table><tbody><tr><td>&amp;nbsp;&amp;micro;CT 35</td><td>Scanco Medical AG</td><td></td><td>Note that Scanco does not offer the&amp;nbsp; &amp;micro;CT 35 anymore. Their smallest scanner is now the&amp;nbsp; &amp;micro;CT 45&amp;nbsp;</td></tr><tr><td>Avizo</td><td>Visualization Sciences Group, VSG</td><td></td><td></td></tr><tr><td>C57BL/6 Mice</td><td>Bioterio Facultad de Ciencias Veterinarias Universidad Nacional de La Plata</td><td></td><td></td></tr><tr><td>Conical tubes</td><td>Daigger</td><td>CH-CI4610-1856</td><td></td></tr><tr><td>Flux cabinet</td><td>Esco</td><td>AC2-458&amp;nbsp;</td><td></td></tr><tr><td>Glass beaker&amp;nbsp;</td><td>Glassco</td><td>GL-229.202.10</td><td></td></tr><tr><td>Glass bottle</td><td>Simax</td><td>CFB017</td><td></td></tr><tr><td>Glass funnel</td><td>HDA</td><td>VI1108</td><td></td></tr><tr><td>HCl</td><td>Carlo Erba</td><td>403872</td><td>Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)</td></tr><tr><td>I&lt;sub&gt;2&lt;/sub&gt;</td><td>Cicarelli</td><td>804211</td><td>When preparing I&lt;sub&gt;2&lt;/sub&gt;KI, manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)</td></tr><tr><td>KI</td><td>Cicarelli</td><td>PA131542.1210</td><td>When preparing I&lt;sub&gt;2&lt;/sub&gt;KI, manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)</td></tr><tr><td>Magnetic stirring</td><td>Arcano</td><td>4925</td><td></td></tr><tr><td>NaOH</td><td>Cicarelli</td><td>1580110</td><td>Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)</td></tr><tr><td>Orbital shaker</td><td>Biomint</td><td>BM021</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Biopack</td><td>2000959400</td><td>Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)</td></tr><tr><td>Paton spatula</td><td>Glassco</td><td>GL-377.303.01</td><td></td></tr><tr><td>PBS</td><td>Biopack</td><td>2000988800</td><td></td></tr><tr><td>Plastic Pasteur pipette</td><td>Daigger</td><td>9153</td><td></td></tr><tr><td>R</td><td>R Project</td><td></td><td>The package geomorph for R was used in the protocol (https://cran.r-project.org/web/packages/geomorph/index.html)</td></tr><tr><td>Scissors&amp;nbsp;</td><td>Belmed</td><td></td><td></td></tr><tr><td>Sodium azide</td><td>Biopack</td><td>2000163500</td><td></td></tr><tr><td>Thermometer</td><td>Daigger</td><td>7650</td><td></td></tr></tbody></table>

micro-ct imaging and morphometric analysis of mouse neonatal brains

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard method for soft tissues, although its low spatial resolution poses some limits for small animals. Here, we describe a protocol for obtaining high-resolution three-dimensional (3D) information on mouse neonate brains and skulls using micro-computed tomography (micro-CT). The protocol includes those steps needed to dissect the samples, stain and scan the brain, and obtain morphometric measurements of the whole organ and regions of interest (ROIs). Image analysis includes the segmentation of structures and the digitization of point coordinates. In sum, this work shows that the combination of micro-CT and Lugol's solution as a contrast agent is a suitable alternative for imaging the perinatal brains of small animals. This imaging workflow has applications in developmental biology, biomedicine, and other sciences interested in assessing the effect of diverse genetic and environmental factors on brain development.

Here, a basic protocol to obtain high-resolution images of neonatal mouse brains is presented. Heads were scanned after immersion in Lugol&#39;s solution. Despite their small size, the main brain anatomical structures, such as the olfactory bulbs, cortex, midbrain, cerebellum, and hindbrain, can be distinguished (Figure 1).
Different analyses can be carried out using these images as inputs. A set of landmarks and semilandmarks in two different anatomical planes we...

Watch this Scientific Journal Video about Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains at JoVE.com

Author Spotlight: High-Resolution Imaging of Mouse Neonate Brains &#8211; A Micro-CT Protocol with Lugol&#39;s Solution Contrast Agent

This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these images.

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard ...

micro-ct-imaging-and-morphometric-analysis-of-mouse-neonatal-brains

Research

JoVE Journal

Neuroscience

1.5K Views.  Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos (ENyS, CONICET-UNAJ-HEC). This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these images.

Video: Author Spotlight: High-Resolution Imaging of Mouse Neonate Brains – A Micro-CT Protocol with Lugol's Solution Contrast Agent

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Developmental Biology

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Medicine

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

Ultrastructural Analysis of a Mouse Brain Section Using Transmission Electron Microscopy

<a href='https://app.jove.com/v/59084/assessment-ultrastructural-neuroplasticity-parameters-after-utero'>Source: Lutz, D., et.al. Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord. J. Vis. Exp. (2019).</a>This video demonstrates the preparation and transmission electron microscopy (TEM) analysis of a brain section from a mouse pup. The protocol starts with osmium tetroxide-fixed tissue embedded in resin, followed by precise trimming to isolate the region of interest and ultramicrotomy to produce semithin and ultrathin sections, which are visualized using light microscopy and TEM, respectively.

Source: Lutz, D., et.al. Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal ...

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Neuroimaging

Developmental & Regenerative Neuroimaging

An Intracerebroventricular Injection to Analyze Murine Cytomegalovirus Distribution in a Neonatal Mouse Brain

Source: Kawasaki, H., et al. <a href="https://app.jove.com/v/54164">Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain.</a> J. Vis. Exp. (2016).
This video demonstrates the intracerebroventricular injection process for studying murine cytomegalovirus (MCMV) distribution in the neonatal mouse brain. MCMV, engineered to express an enhanced green fluorescent protein (EGFP) for visibility, is injected into the lateral ventricle of a mouse pup&#39;s brain. The injected virus circulates through the cerebrospinal fluid, infecting cells in the periventricular region, including the marginal zone, choroid plexus, and subventricular zone.

1. ICV Injection of MCMV and Fluorescent Microbeads into Neonatal Mice

	Maintain normal pregnant ICR mice in a temperature-controlled facility under a 12 ...

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

Summary

Explore More Videos

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Ultrastructural Analysis of a Mouse Brain Section Using Transmission Electron Microscopy

An Intracerebroventricular Injection to Analyze Murine Cytomegalovirus Distribution in a Neonatal Mouse Brain