RVS is supported by new investigator start-up funds from Albert Einstein College of Medicine of Yeshiva University.

1. Animal Perfusion and Cerebellum Dissection
<ol>
 <li>Depending on the protein, perfusion may be essential for successful staining1,2. Transcardiac perfusion is an invasive, non-survival procedure that requires the proper use of anesthetics. Correct training, institutional approval, and IACUC approval are all necessary before attempting the procedure. It is always a good idea to consult the institution's veterinarians to get help in identifying experimental requirements and acquiring the correct training. Before the procedure, the animal should be deeply anesthetized and not responsive to stimuli such as foot or tail pinch. Using scissors make a cut in the abdominal skin and muscle wall and then continue to open the rib cage by cutting just adjacent to the sternum on each side. In addition, the experimenter can create a larger internal working space by cutting the fascia surrounding the diaphragm. By lifting the abdominal and thoracic wall superiorly, the heart will now be exposed. Make a small cut in the right atrium to allow blood to flow out and immediately insert the perfusion needle into the left ventricle. A basic hydrostatic pump is preferred to deliver the perfusion solutions (see Table 1 for details). First, flush the blood out with 0.1M phosphate buffered saline (PBS, see Table 3) until the fluid runs clear. Then, turn off the hydrostatic pump, quickly switch the solution delivery tube to a container of 4% paraformaldehyde (PFA, see Table 3) diluted in PBS and then turn the pump back on to deliver the fixative. The use of a pump is not necessary: with practice, two syringes filled with the PBS and 4% PFA can be used to slowly deliver the solutions.</li>
 <li>It is critical to carefully dissect the brain out of the skull without introducing any lesions to the tissue. Even small lacerations to the brain during dissection will gradually expand into visible crevices that trap antibodies and cause artifactual staining during the processing of the tissue (e.g. red asterisks in Fig 2). For cerebellar dissections, it is typical to cut the skin down the middle of the head and then gently tease the flaps apart to expose the skull. Cut the skull from anterior to posterior along the dorsal midline, and also laterally along both sides of the skull. Do not cut beyond the bregma as this runs the risk of nicking the cerebellum. Using forceps gently lift the front of the skull away from the brain being careful not to tear away brain tissue that is attached to the meninges. Sever the cranial nerves on the ventral side of the brain.</li>
 <li>Separation of the cerebellum from the rest of the brain is important for two reasons. First, it allows for exploration of expression patterns in the anterior lobules, which are hidden from view by the colliculi in situ. The second is that separating the tissue into smaller, isolated regions allows for more complete fixation, which can facilitate the staining of some proteins. Throughout this final dissection step, care should be taken to not touch the cerebellum with the tips of the forceps. Insert the tips of a pair of forceps into the middle of the superior and inferior colliculi. With a second set of forceps slowly tease away the inferior colliculi from the cerebellum. Then, lay the cerebellum so that its anterior side is facing down and the brainstem is pointing upwards. Insert the forceps into the fourth ventricle between the brainstem and lobules IX/X of the cerebellum. Cut the peduncles by pinching with the forceps on both sides and then gently lift the cerebellum away from the brainstem. Once the cerebellum is isolated, post-fix it by immersion in 4% PFA at 4 &deg;C for a minimum of 24-48 hours. The cerebellum can be stored in 4% PFA for an extended period of time. The meninges should be removed either before or after staining for better visibility of the expression patterns. If the experimenter chooses to remove the meninges before staining there is a strong possibility that the cerebellum will be nicked during the process. Peeling away the meninges at the end of the staining procedure is much simpler because they become easier to locate after they acquire weak background staining and become frail post-processing.</li>
</ol>
2. Processing the Tissue for Wholemount Staining
Because the wholemount staining approach takes longer than immunohistochemical staining of tissue sections, it is helpful to plan the timeline for each of the experiments as shown in the example calendar provided (Table 2). Before starting, there are several key things to keep in mind. 1) The microtubes containing the tissue should be rotated on a nutator at all times, except during the freeze/thaw process. 2) Several solutions must be made fresh for each experiment (See Table 3 for solution recipes). 3) Throughout the protocol, when changing solutions, gently pour out the spent solution rather than removing the cerebellum with forceps, to avoid touching the cerebellum. Then, after mixing the new solution in another container, use a pipette to gently add the fresh solution to the tube.
2.1 Day 1:
<ol class="lalpha">
 <li>Fix the cerebellum in Dent's fix3 at room temperature for 6-8 hours rocking gently on the nutator. If methanol is destructive to your antigen, consider replacing steps 2.1a through 2.3a with an antigen retrieval method. The heat and buffers used for antigen retrieval will help prepare the tissue for antibody penetration.</li>
 <li>Bleach the cerebellum in Dent's Bleach3 overnight at 4 &deg;C.</li>
</ol>
2.2 Day 2:
<ol class="lalpha">
 <li>Dehydrate the cerebellum in two rounds of 100% MeOH at room temperature for 30 min each.</li>
 <li>Then, to enhance the penetration of the solutions, subject the tissue to a series of freeze/thaw cycles. Carefully place the tube in a container with dry ice and freeze for 30 min. Then, remove the tube and leave it at room temperature on the bench for 15 minute. The freeze/thaw step should be performed four times (at least).</li>
 <li>After the last thawing of the tissue, place the tube into a -80 &deg;C freezer overnight.</li>
 <li>The tissue can be stored for prolonged periods of time at -80 &deg;C either immediately before or after the freeze/thaw steps. Otherwise, the protocol should be continued.</li>
</ol>
2.3 Day 3:
<ol class="lalpha">
 <li>Rehydrate the cerebellum by washing it in 50% MeOH/50% PBS, 15% MeOH/85% PBS, and 100% PBS for 60-90 minutes each at room temperature.</li>
 <li>Next, for adult mouse cerebella, subject the tissue to enzymatic digestion with 10&mu;g/ml Proteinase K in PBS for 2-3 minutes. No digestion step is necessary for cerebella of young animals (P5 or younger in mouse). Longer digestion is suggested for larger brains. For instance, digestion could be as high as 5-6 minutes for primate brains4.</li>
 <li>After digestion, immediately wash the cerebellum in PBS three times for 10 minutes each at room temperature to remove the Proteinase K.</li>
 <li>Block the tissue in PMT (see Table 3) overnight at 4 &deg;C. Milk powder is often the first choice when selecting a blocking agent for protein work because it is inexpensive, effectively lowers background staining, and typically does not interfere with antibody binding or access to the antigen. Animal serums can also be used, although the experimenter should be aware of the added cost and must first test the serum quality for cross-reactive immunoglobulins and ability to produce an optimal signal to noise ratio.</li>
</ol>
2.4 Day 4-5:
<ol class="lalpha">
 <li>Incubate the tissue in primary antibodies diluted in PMT supplemented with 5% DMSO for 48 hours at 4 &deg;C (larger cerebella will require increased incubation times).</li>
</ol>
2.5 Day 6:
<ol class="lalpha">
 <li>Wash the cerebellum 2-3 times for 2-3 hours each in PMT at 4 &deg;C to remove unbound primary antibodies.</li>
 <li>Then, incubate the tissue in secondary antibodies in a solution containing PMT with 5% DMSO at 4 &deg;C overnight (larger cerebella may require increased incubation times).</li>
</ol>
2.6 Day 7:
<ol class="lalpha">
 <li>Wash the cerebellum 2-3 times for 2-3 hours each in PMT at 4 &deg;C.</li>
 <li>Give the tissue a final wash with PBT (see Table 3) for 1-2 hours. This step removes excess milk and enhances the clarity of the staining.</li>
 <li>Finally, incubate the tissue in DAB (3,3'-diaminobenzidine) solution until the optimal staining intensity is reached. The brown reaction product should be clearly visible within ~10 minutes. It is best to use DAB in the dark as light causes the chromogen to precipitate, which can increase background staining.</li>
</ol>
2.7
When the optimal staining intensity is reached, stop the reaction by placing the cerebellum in PBS with 0.04% sodium azide. The tissue may be stored long-term in this solution. Sodium azide is a potent inhibitor of bacterial growth but should be avoided until this step as it also inhibits horseradish peroxidase.
2.8 Optional amplification procedure:
follow normal protocol but these steps should be performed in place of steps 2.5b-2.7 above.
<ol class="lalpha">
 <li>Incubate the tissue overnight at 4 &deg;C in biotin-conjugated secondary antibodies in PBST (see Table 3) with 5% DMSO.</li>
 <li>Rinse the tissue for 2-3 hours each in 3-4 changes of PBST at room temperature.</li>
 <li>Incubate the cerebellum overnight at 4 &deg;C in ABC complex solution in PBST.</li>
 <li>Rinse the tissue for 2-3 hours each at room temperature in 3-4 changes of PBS.</li>
 <li>Incubate the tissue in freshly-prepared DAB solution, as described above.</li>
 <li>When the staining is optimal, stop the reaction by washing the cerebellum in PBS with 0.04% sodium azide.</li>
</ol>
3. Imaging the Stained Tissue
<ol>
 <li>Wholemount images can be captured by using, for instance, a Leica DFC3000 FX camera mounted on a Leica MZ16 FA stereomicroscope running Leica Application Suite FX software. If desired, deconvolution microscopy can be used to acquire multiple consecutive images in different focal planes, with each image having only a single lobule in crisp focus. Then, the images can be compressed into a single stack and software rendered to remove distortion. The final composite image will illustrate cerebellar surface expression patterns with all visible patterning in clear focus.</li>
 <li>Wholemount stained tissue should be imaged while immersed in PBS (or another medium that will give an optimal refractive index). In order to easily position the wholemount for imaging, make a 1% agar gel in a plastic Petri dish. Cut small holes into the gel. The holes will allow the cerebellum to be wedged into the desired orientation.</li>
 <li>Raw data are imported into Adobe Photoshop CS4 and adjusted for contrast and brightness levels.</li>
</ol>
Animals
All animal studies were carried out under an approved IACUC animal protocol according to the institutional guidelines at Albert Einstein College of Medicine. Male and female outbred Swiss Webster (Taconic, Albany, NY) mice were maintained in our colony and used for all studies. Euthanized adult rats were kindly provided by Dr. Bryen Jordan (Albert Einstein College of Medicine). All animals were at least one month old.
4. Representative Results
The cerebellum is compartmentalized by molecular expression into four transverse zones: the anterior zone (AZ: ~lobules I-V), the central zone (CZ: ~lobules VI-VII), the posterior zone (PZ: ~lobules VIII-dorsal IX) and the nodular zone (NZ: ~lobules IX ventral and X)5. Each zone contains a unique array of parasagittal stripes1,2,5,6 (Fig. 1). ZebrinII expression in Purkinje cells reveals stripes in the AZ and PZ (Fig. 2) and uniform expression in the CZ and NZ (Fig. 2). The parasagittal organization of Purkinje cells is mirrored by the terminal field topography of afferent fibers. Cocaine- and amphetamine-regulated transcript (CART) peptide is expressed in subsets of climbing fibers (Fig. 3a) that project to stripes of Purkinje cell dendrites in the molecular layer of the cerebellar cortex7 (Fig. 3b). With the appropriate modifications, such as amplification7, the wholemount protocol allows for the visualization of olivocerebellar patterns without the need for a laborious, time consuming reconstructions from staining tissue sections (Fig. 3b).
<img alt="Figure 1" src="/files/ftp_upload/4042/4042fig1.jpg" /> 
Figure 1. A. ZebrinII/aldolaseC expression reveals sagittal bands in transverse sections of the cerebellum. Scale bar = 500 &mu;m. B. ZebrinII/aldolaseC is expressed exclusively in Purkinje cell somata and dendrites. Scale bar = 150&mu;m (gl = granular layer; pcl = Purkinje cell layer; ml=molecular layer).
<img alt="Figure 2" src="/files/ftp_upload/4042/4042fig2.jpg" /> 
Figure 2. A wholemount cerebellum stained for ZebrinII/aldolaseC showing Purkinje cell stripes in images of the anterior (AZ), central (CZ), and posterior (PZ) zones of the cerebellum. Here we also show an example of the negative consequence of nicking the cerebellum. The resulting artifactual staining is indicated with red asterisks. Scale bar = 1 mm (LS = lobulus simplex; PML = paramedian lobule; COP = copula pyramidis).
<img alt="Figure 3" src="/files/ftp_upload/4042/4042fig3.jpg" />
 
Figure 3. A. CART is expressed in climbing fiber terminals in the molecular layer. Scale bar = 100 &mu;m (ml = molecular layer; pcl = Purkinje cell layer; gl = granular layer). B. Wholemount immunohistochemistry of CART expression in the NZ. Scale bar = 2 mm (COP = copula pyramidis; PML = paramedian lobule; PFL = paraflocculus; FL = flocculus).

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We have nothing to disclose.

We have described the technical details required for successful wholemount staining using a versatile immunohistochemical approach for revealing protein expression in the developing and adult brain. By using this approach, complex molecular expression patterns can be analyzed and brain topography appreciated without the need for laborious and time consuming tissue sectioning procedures.
This protocol has been used to reveal the patterned expression of several Purkinje cell proteins in both the adult1,2,8,9 and early postnatal mouse cerebellum10,11,12. Our original studies also demonstrated the utility of the wholemount approach for examining granule cell antigen expression1 and anterogradely traced mossy fibers2, which reside ~250 &mu;m below the pial surface in granular layer. In addition, several studies have made modifications to the original protocol for successful staining of proteins in Purkinje cells1,13 (antigen retrieval) and afferent fibers7,14 (signal amplification; Fig. 2). Moreover, wholemount immunohistochemical staining has also been applied to the cerebella of multiple mammalian4,15 and avian16,17 species, used to map patterned Purkinje cell loss in mouse models of neurodegenerative diseases18,19, and modified to stain heart20, lung21, cranial nerves22 and corneal nerves23. The major departure of the current protocol from previous descriptions of the wholemount method1,2 is that here we provide not only updated details for better success when using the procedure, but also a complete guide for fixing the tissue, dissecting the tissue and an in depth video illustration of the technique.
 
There are several limitations of wholemount staining. First, only surface patterns are typically visualized without further dissection and staining or without increasing the length of time that the tissue is incubated in antibodies. Further, staining will not reach deep within the cortex if it is hidden due to foliation1,2. However, wholemount stained tissue can be sectioned after processing and restained, with the same antibody or another, to reveal cellular expression profiles deeper within the tissue1,2. Second, the basic wholemount staining protocol is long. We suggest that each experimenter empirically determine the staining efficiency of their antibodies and shorten the post-fixation time, shorten the number and length of washes, and/or shorten the length of incubation in the primary antibodies. With these adjustments the protocol may be completed in a considerably shorter time. Third, for large cerebella the wholemount staining protocol requires large volumes of the antibody solutions, which can be expensive and/or only available in limited quantities. However, it is possible to save the primary antibodies after each run by freezing the solution at -20&deg;C. Each experimenter will have to determine whether their antibody can withstand freezing before it is re-used. We have successfully stained tissue using previously frozen aliquots of zebrinII. Fourth, not all antibodies are compatible with the basic wholemount approach. For example, the antibody commonly used to detect the striped expression of the small heat shock protein, HSP25, requires that the tissue be first processed for antigen retrieval13. Regardless, as for staining tissue sections, traditional methods of troubleshooting antibody-antigen binding have also proven useful for optimizing wholemount staining.
We are currently exploring the possibility of adapting our wholemount protocol for use with fluorescent-tagged secondary antibodies. With the appropriate microscope equipped with the ability to image different colored fluorophores under low-power, one would be able to not only analyze the organization of multiple Purkinje cell maps in the same animal, but also examine the relationship between Purkinje cell stripe patterns and the topography of WGA-Alexa labeled afferent fibers in three-dimensions2,4.7,24. Furthermore, the recent availability of large scale gene expression databases (Allen Brain Atlas, Genepaint, Brain Gene Expression Map) have opened new avenues for merging our high throughput wholemount approach with whole genome analyses for examining the molecular topography of the entire brain.

<table border="1">
<tbody>
 <tr>
 <td>Materials </td>
 <td>Function in protocol</td>
 </tr>
 <tr>
 <td>Perfusion pump (Fisher Scientific/13-876-2)</td>
 <td>Allows for consistent and slow perfusion. </td>
 </tr>
 <tr>
 <td>Sharp-tip Scissors (FST/14081-08)</td>
 <td>General use in perfusion and dissection.</td>
 </tr>
 <tr>
 <td>Blunt-tip Forceps (FST/91100-12)</td>
 <td>To stabilize the heart for insertion of the perfusion needle.</td>
 </tr>
 <tr>
 <td>Forceps (FST by Dumont AA/11210-10)</td>
 <td>For use during dissection of the brain from the skull and to separate the cerebellum from the rest of the brain. These are essential because they have a slightly rounded tip that helps minimize damage to the cerebellum during dissection. </td>
 </tr>
 <tr>
 <td>Nutator (Fisher Scientific)</td>
 <td>Used to keep tissue in motion during incubation periods.&nbsp; </td>
 </tr>
 <tr>
 <td>1.5 mL tube (Sarstedt/Screw Cap Micro Tube)</td>
 <td>All steps of the histochemistry protocol take place in these microtubes. The rounded bottom ensures that the cerebellum stays in motion.&nbsp; </td>
 </tr>
 <tr>
 <td>Perforated spoon (FST/10370-17)</td>
 <td>Used to keep wholemounts in the microtubes while gently decanting out the spent solution. </td>
 </tr>
 <tr>
 <td>Leica MZ16 FA microscope</td>
 <td>Used to examine wholemount staining.</td>
 </tr>
 <tr>
 <td>Leica DFC3000 FX camera</td>
 <td>Used to capture wholemount images. </td>
 </tr>
 </tbody>
</table>
Table 1.
<table border="1">
<tbody>
 <tr>
 <td colspan="9">Example calendar for a typical wholemount experiment</td>
 </tr>
 <tr>
 <td>Day 1</td>
 <td colspan="4">Dent's fix, room temperature, 8 hrs</td>
 <td colspan="4">Dent's bleach, 4&deg;C, overnight</td>
 </tr>
 <tr>
 <td>Day 2</td>
 <td colspan="3">100% MeOH, room temperature, 2x, 30 min each</td>
 <td colspan="3">100% MeOH, Freeze/thaw, 
 4x, 30 min/15 min</td>
 <td colspan="2">100% MeOH, -80&deg;C, overnight</td>
 </tr>
 <tr>
 <td>Day 3</td>
 <td>50% MeOH/50% PBS, room temperature, 60-90 min</td>
 <td colspan="2">15% MeOH/ 85% PBS, room temperature, 60-90 min</td>
 <td colspan="2">100% PBS, room temperature, 60-90 min</td>
 <td>10&mu;g/mL Proteinase K in PBS, room temperature, 2-3 min</td>
 <td>100% PBS, room temperature, 3x, 10 min each</td>
 <td>PMT, 4&deg;C, overnight</td>
 </tr>
 <tr>
 <td>Day 4-5</td>
 <td colspan="8">PMT + 1&deg; antibody + 5% DMSO, 4&deg;C, 48 hrs </td>
 </tr>
 <tr>
 <td>Day 6</td>
 <td colspan="3">PMT, 4&deg;C, 2-3x, 2-3 hrs each</td>
 <td colspan="5">PMT + 2&deg; antibody + 5% DMSO, 4&deg;C, 24 hours (Or begin amplification steps with ABC complex)</td>
 </tr>
 <tr>
 <td>Day 7</td>
 <td colspan="2">PMT, 4&deg;C, 2-3x, 2-3 hrs each</td>
 <td colspan="3">PBT, room temperature, 2 hrs</td>
 <td colspan="3">Incubate in fresh DAB in PBS until optimal staining is visualized</td>
 </tr>
</tbody>
</table>
Table 2.
<table border="1">
<tbody>
 <tr>
 <td colspan="2">Recipes (*=prepare fresh every time)</td>
 </tr>
 <tr>
 <td>PBS (phosphate buffered saline)</td>
 <td>0.1M phosphate buffered saline in deionized water. pH 7.2 (Sigma tablets; P4417)</td>
 </tr>
 <tr>
 <td>PFA (Paraformaldehyde)</td>
 <td>Made and stored frozen as a 20% solution and then diluted to 4% in PBS for the working solution (Fisher Scientific; T353)</td>
 </tr>
 <tr>
 <td>Dent's Fixative3*</td>
 <td>4 parts methanol 
 1 part dimethylsulfoxide (DMSO; Fisher Scientific; D159-4)</td>
 </tr>
 <tr>
 <td>Dent's Bleach3*</td>
 <td>4 parts methanol 
 1 part dimethylsulfoxide (DMSO; Fisher Scientific; D159-4) 
 1 part 30% hydrogen peroxide </td>
 </tr>
 <tr>
 <td>Enzymatic Digestion </td>
 <td>10 &mu;g/ml of Proteinase K (Roche Diagnostics; 03115828001) in PBS. </td>
 </tr>
 <tr>
 <td>PBST</td>
 <td>PBS containing: 
 0.1% Tween-20 (Fisher Scientific, BP337; Triton can also be used in place of Tween-20 in all instances.)</td>
 </tr>
 <tr>
 <td>PMT25*</td>
 <td>PBS containing: 
 2% nonfat skim milk powder (Carnation preferred) 
 0.1% Tween-20 (Fisher Scientific; BP337)</td>
 </tr>
 <tr>
 <td>PBT25*</td>
 <td>PBS containing: 
 0.2% bovine serum albumin (Sigma; B9001S) 
 0.1% Tween-20 (Fisher Scientific; BP337)</td>
 </tr>
 <tr>
 <td>DAB*</td>
 <td>Dissolve one 10-mg tablet of 3,3-diaminobenzidine (Sigma-Aldrich; D5905) in 40 ml of PBS. Add 10 &mu;l of 30% hydrogen peroxide to initiate reaction). </td>
 </tr>
 <tr>
 <td>ABC Complex Solution</td>
 <td>Vectastain kit (Vector laboratories, Inc; PK-4000)</td>
 </tr>
 </tbody>
</table>
Table 3.

wholemount immunohistochemistry for revealing complex brain topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Watch this Scientific Journal Video about Wholemount Immunohistochemistry for Revealing Complex Brain Topography at JoVE.com

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

wholemount-immunohistochemistry-for-revealing-complex-brain-topography

Nanyang Technological University

Research

JoVE Journal

Neuroscience

15.5K Views.  Albert Einstein College of Medicine, Yeshiva University. Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

Video: Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Revealing Neural Circuit Topography in Multi-Color

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.

We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Purification of the Dendritic Filopodia-rich Fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

Summary

Explore More Videos

Chapters in this video

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

Revealing Neural Circuit Topography in Multi-Color

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Purification of the Dendritic Filopodia-rich Fraction

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Assessment of Long-term Depression Induction in Adult Cerebellar Slices