This work was supported by the Labex MemoLife and by the ARSEP (Fondation pour l&#8217;aide &#224; la recherche sur la scl&#233;rose en plaques)

1. Solutions and Materials &#160;
<ol>
	<li>Prepare isolation vessel solutions: B1, add 1.5 ml of HEPES 1M to 150 ml of HBSS; B2, add 3.6 g of Dextran to 20 ml of B1; B3, add 1 g of BSA to 100 ml of B1.</li>
	<li>Modify the filter holder by cutting the bottom off the upper screwing part.</li>
	<li>Prepare immunostaining solutions: Fixation solution, 4% Paraformaldehyde in PBS pH 7.4; Permeabilization/blocking solution, dilute goat serum to 5% and Triton X100 to 0.25% in PBS pH 7.4. 
	Note: Fixation depends on the type of primary antibodies used.</li>
</ol>
2. Dissection
Note: Sterile conditions are not required, unless vessels are used for cell culture purposes.
<ol>
	<li>Prepare a 150 ml beaker with 20 ml of B1. Keep on ice and cover with parafilm to avoid air contamination.</li>
	<li>Deeply anesthetize the mouse under a hood with a small paper towel soaked in 1 ml of pure Isoflurane that is added into the cage. Anesthesia is verified by a lack of reaction to a toe pinch. Kill the mouse by cervical dislocation. 
	NOTE: These steps are accomplished in compliance with national and institutional regulations.</li>
	<li>Optional: Perform intracardiac perfusion with 20 ml of PBS 1x&#160;to eliminate the blood content24.</li>
	<li>Section the skin with a scalpel from the neck to the nose and pull it away. Remove all contaminating hairs with PBS 1x.</li>
	<li>To open the skull, first insert scissors anteriorly to the olfactory bulb, and open the scissors to rupture the skull in two parts.</li>
	<li>Carefully remove the brain using a brain spatula. Dissect out the choroid plexus from the lateral ventricles as they would contaminate the blood vessel preparation38. 
	NOTE: Optional: The final preparation will contain parenchymal and meninges vessels. If not desired, meninges can be peeled off following the procedure described by&#160;38.</li>
	<li>Transfer the brain into the beaker containing B1 solution on ice. Up to 8 brains can be treated together.</li>
</ol>
3. Brain Tissue Homogenization
<ol>
	<li>Using two scalpels, manually and vigorously beat the brain in the B1 solution subsequently obtaining small pieces of about 2 mm.</li>
	<li>Homogenize the preparation with an automatized Dounce homogenizer, performing 20 strokes at 400 rpm. Ensure that the glass tube is maintained in ice and that the upper part of the douncer is in solution when moving up and down, so as to prevent the formation of air pockets. If several samples are prepared, wash the douncer with ionized water between each homogenization.</li>
</ol>
4. Vessel Purification
<ol>
	<li>Transfer the homogenate into a 50 ml plastic tube and proceed to the centrifugation at 2,000 g for 10 min at 4 &#176;C. A large white interface (mostly myelin) will form on the top of the vessel pellet (red if no perfusion was performed) .</li>
	<li>Discard the supernatant. The vessel pellet and the white interface will remain attached together. Add 20 ml of ice-cold B2 solution and shake the tube manually and vigorously for 1 min.</li>
	<li>Proceed to the second centrifugation at 4,400 g for 15 min at 4 &#176;C. The myelin will now form a dense white layer at the surface of the supernatant.</li>
	<li>Carefully detach the myelin layer from the tube walls by holding the tube and slowly rotating it to allow the supernatant to pass along the walls. Discard myelin with the supernatant. The pellet containing the vessels remains attached at the bottom of the tube.</li>
	<li>Blot the inside wall of the tube with an absorbent paper wrapped around a 5 ml plastic pipette and remove all residual fluids, avoid touching the vessel pellet. Keep the tube upside-down on an absorbent paper to drain any remaining liquid.</li>
	<li>Suspend the pellet in 1 ml of ice-cold B3 solution by pipetting up and down with low-binding tips , keeping the tube on ice, then add another 5 ml of B3 solution. Make sure that vessels are dispersed as much as possible and do not form aggregates.</li>
</ol>
5. Filtration
<ol>
	<li>Prepare a beaker on ice with 30 ml of ice-cold B3 solution. Cover with parafilm to avoid air contamination.</li>
	<li>Place a 20 &#181;m-mesh filter on a modified filter holder on the top of a becker&#160;flask and equilibrate by applying 10 ml of ice-cold B3 solution.</li>
	<li>Pour the vessel preparation on the filter and rinse the vessels with 40 ml of ice-cold B3 solution.</li>
	<li>Recover the filter using clean forceps and immediately immerse it in the beaker containing the B3 solution. Detach the vessels from the filter by shaking it gently.</li>
	<li>Pour the beaker content in a 50 ml plastic tube and centrifuge at 2,000 g for 5 min at 4 &#176;C.</li>
	<li>Note: Alternatively, the brain vessels&#8217; suspension from step 4.6 can be filtered onto a 100&#160;&#181;m-mesh filter. In this case, larger vessels are preferentially retained on the filter while the flow-through contains microvessels (mainly capillaries), which are then filtered on a 20 &#181;m-mesh filter as above.</li>
	<li>Resuspend the pellet of microvessels in 1 ml of ice-cold B3 solution and transfer it by pipetting it into a 1.5 ml Eppendorf tube. Centrifuge at 2,000 g for 5 min at 4 &#176;C.</li>
</ol>
6. Fixation, Permeabilization and Blocking
<ol>
	<li>Transfer vessels by pipetting into 0.2 ml PCR tubes containing PBS using a 1.0 ml low binding tip. Be careful not touching the vessels as they may adhere to the outside part of the tip. Perform all the following steps under a binocular microscope.</li>
	<li>For each of the following medium changes, leave the tubes on ice until the vessels have reached the bottom, and pipette out most of the liquids using long and thing gel-loading pipet tips.</li>
	<li>Remove most of the PBS and add 200 &#181;l of fixative solution. Suspend the vessels by shaking gently and incubate for 20 min at RT.</li>
	<li>Pipette out the fixative solution and replace it with 200 &#181;l of 1x&#160;PBS&#160;(first wash), incubate for 5 min at RT and repeat this step 3 times. Each time, verify that vessels have correctly sunk to the bottom of the tubes and pipette out 1x&#160;PBS, ensuring the vessels are not touched.</li>
	<li>After the last wash, replace 1x&#160;PBS by the permeabilization/blocking solution and incubate 1 hr at RT, resuspending the vessels by shaking gently from time to time.</li>
</ol>
7. Immunostaining
<ol>
	<li>Replace the permeabilization/blocking solution with the mix of primary antibodies (see references of the antibodies used here and dilutions in the Materials template table) diluted in the permeabilization/blocking solution, and incubate at 4 &#176;C O/N.</li>
	<li>After 3 washes in PBS at RT, incubate the vessels with the mix of secondary antibodies diluted in PBS for 2 hr at RT.</li>
	<li>Note: we strongly recommend performing nuclear staining with Hoechst (1:2,000) or DAPI (1:2,000) in order to distinguish vessels from possible contaminating hairs or dusts under the microscope.</li>
	<li>After 3 washes in PBS, resuspend the vessels in 50 &#181;l of PBS.</li>
</ol>
8. Mounting and Observation
<ol>
	<li>Prepare a tip with a siliconized glass capillary: cut a P200 pipette tip using a scalpel and adjust the capillary inside. The approximate volume of the capillary is 40 &#956;l.</li>
	<li>Transfer the vessels to a glass slide using the siliconized glass capillary and carefully remove the liquid with a piece of absorbent paper.</li>
	<li>Apply a single drop of mounting medium onto the vessels and hold a coverslip at 45&#186; allowing the drop to spread along the edge of the slip. Let go off the coverslip and allow medium to spread slowly. Let it dry O/N&#160;at RT with protection from light. Vessels can be observed under a fluorescent confocal microscope.</li>
</ol>

The authors have nothing to disclose.

The blood&#8211;brain barrier regulates the passage of physiological substances in and out of the CNS and protects it against potentially harmful substances present in the blood. It is involved in several CNS pathologies, including neurodegenerative diseases2 and brain tumors28. The extremely low permeability of the BBB hampers also the passage of therapeutic agents targeting neural cells and the development of methods intending to reversibly open the BBB with no deleterious consequences for the bra...

Proper function of the central nervous system (CNS) requires a highly regulated extracellular environment, and its metabolic demands are huge compared to other organs1. The CNS is also extremely sensitive to a wide range of chemicals, generally harmless to peripheral organs but to it, neurotoxic. To ensure correct functioning, most of the CNS&#39; vasculature forms an endothelial barrier; the blood-brain barrier (BBB), which controls the flow of molecules and ions as well as the passage of immune cells between the blood and the brain, thereby maintaining proper homeostasis2, but also limiting the entry of therapeutic drugs, thus hampering treatments of neurological disorders3. At the cellular level, the BBB is mainly sustained by extensive tight junctions between endothelial cells, polarized expression of efflux transporters and a very low transcytosis rate4. Properties and functions of the BBB are mostly induced by neighboring cells4. In particular, pericytes play an important role in inducing and maintaining the BBB5,6. Being contractile cells, pericytes also regulate blood flow7 as do the smooth muscle cells surrounding large vessels. Finally, astrocytes, the major glial cells of the brain, send large processes named endfeet around most of the brain vasculature8 and modulate BBB integrity and immune quiescence9, the transfer of metabolites to neurons10, and induce the tight coupling between neuronal activity and blood flow11,12.
The ability to study the molecular and cellular properties of the cerebrovascular system is crucial to characterize better its contribution to brain physiology and physiopathology. To tackle this question, strategies to isolate the brain&#39;s cerebrovascular system have been developed, which allow for the preparation of intact brain vessel fragments. Cerebral vessel purification was initially described using bovine brains13 and improved and adapted to other species, in particular rodents14. In this last study, the use of filters of varying size was introduced to separate brain vessels in to fractions enriched in vessels of different diameters. Interestingly, in such preparations, endothelial cells kept their metabolic properties15, transporter functionality16 and polarization17. Here, we describe in detail this protocol and further demonstrate that isolated vessels retain most of their in situ structures. Endothelial cells remain linked by tight junctions and surrounded by a continuous basal lamina. Pericytes and smooth muscle cells remain attached to the endothelial layer, as well as perivascular astrocyte membranes. However, astrocytes, microglial cells, neurons and oligodendrocytes are eliminated. Lastly, we describe a procedure to perform immunostaining on isolated brain vessels.
Until now most of the molecular and cellular studies concerning the cerebrovascular system have been performed on purified brain vessel cells dissociated by cell-sorting using cell specific reporter mouse strains or immunostaining-based procedures18,19. Although these techniques allows for the isolation of almost pure cerebrovascular cell populations, isolated cells completely lose their in situ morphology and interactions, which in turn, greatly affects their molecular and cellular properties. The protocol described here, allowing for the isolation of whole cerebrovascular fragments with no need of specific antibodies or transgenic mouse stains, offers a good alternative as the overall structure of isolated cerebral vessels is conserved, thus, lessening repercussions on their molecular properties. Isolated vessels might then be used for studying gene activity, protein synthesis and regulation at the BBB as recently described20,21. Finally, compared to laser capture microdissection22,23&#160;the present protocol is inexpensive, easy to perform and rapidly adaptable in to any laboratory.

<table border="0">
	<tbody>
		<tr>
			<td>Tissue Grinder Size C</td>
			<td>Thomas scientific</td>
			<td>3431E25</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge 5415&#160;R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td>5811000320</td>
			<td></td>
		</tr>
		<tr>
			<td>High-performance, Modular Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact System Provides High Quality Leica LED1000</td>
			<td>Leica</td>
			<td>LED1000</td>
			<td></td>
		</tr>
		<tr>
			<td>low binding tips (P1000)</td>
			<td>Sorenson BioScience</td>
			<td>14200T</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinnex 47mm filter holder PP 8/Pk</td>
			<td>Millipore</td>
			<td><a href="http://www.millipore.com/catalogue/itemdetail.do?id=SX0004700">SX0004700</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon net filter disc Hydrophilic 20&#181;m 47mm 100/Pk</td>
			<td>Millipore</td>
			<td><a href="http://www.millipore.com/catalogue/itemdetail.do?id=NY2004700">NY2004700</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon net filter disc Hydrophilic 100&#181;m 47mm 100/Pk</td>
			<td>Millipore</td>
			<td><a href="http://www.millipore.com/catalogue/itemdetail.do?id=NY1H04700">NY1H04700</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Wall Borosilicate Tubing</td>
			<td>Sutter Instrument</td>
			<td>B150-86-7.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Thermo Scientific</td>
			<td>1014356290F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slips, Thickness 1</td>
			<td>Thermo Scientific</td>
			<td>P10143263NR1</td>
			<td></td>
		</tr>
		<tr>
			<td>0,2 ml Thin-walled tubes and domed cap</td>
			<td>Thermo Scientific</td>
			<td>AB-0266</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;PARAFILM&#174; M (roll size 4 in. &#215; 125 ft)</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Life technology</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/14175129">14175-129</a></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Life technology</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran from Leuconostoc spp. Mr ~70,000</td>
			<td>Sigma</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 10X</td>
			<td>Euromedex</td>
			<td>ET330</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Formaldehyde (w/v), Methanol-free&#160;</td>
			<td>Thermo Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#8482; X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>bisBenzimide H 33342 trihydrochloride (Hoechst)</td>
			<td>Sigma</td>
			<td>14533</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor&#174; 488 Conjugate; Dilution 1/100</td>
			<td>Life technology</td>
			<td>I21411</td>
			<td></td>
		</tr>
		<tr>
			<td>Agrin (rabbit) ; dilution 1/400</td>
			<td colspan="2">kindly provided by Dr Markus A Ruegg</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti ZO-1 (mouse, clone 1A12)</td>
			<td>Life technology</td>
			<td>33-9100</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti Smooth Muscle Actin (mouse, clone 1A4)</td>
			<td>Sigma</td>
			<td>A2547</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti GFAP (mouse, clone GA5)</td>
			<td>Sigma</td>
			<td>G3893</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti AQP4 (rabbit)</td>
			<td>Sigma</td>
			<td>A5971</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti Cx43 (mouse, Clone&#160; 2)</td>
			<td>BD Biosciences</td>
			<td>610061</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti Olig2 (rabbit)</td>
			<td>Millipore</td>
			<td>AB9610</td>
			<td>&#160;dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti NF-M (mouse)</td>
			<td colspan="2">provided by Dr Beat M. Riederer, University of Lausanne, Switzerland.</td>
			<td>&#160;dilution 1:10</td>
		</tr>
		<tr>
			<td>Anti Iba1 (rabbit)</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td>&#160;dilution 1:400</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 488 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td>Life technology</td>
			<td>A11029</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 488 conjugate</td>
			<td>Life technology</td>
			<td>A11034</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 555 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td>Life technology</td>
			<td>A21424</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 555 conjugate</td>
			<td>Life technology</td>
			<td>A21429</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
	</tbody>
</table>

purification of mouse brain vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

Here, we describe a protocol allowing for the mechanical isolation of brain vessels14. Figure 1 summarizes the main steps of this technique. The architecture of brain vessels is complex and includes several cell types, i.e., endothelial cells sealed by tight junctions and surrounded by pericytes, smooth muscle cells, and astrocyte foot processes9. Thus, following isolation of brain vessels, we aimed to characterize the structure of purified vessels by immunostaining as desc...

Watch this Scientific Journal Video about Purification of Mouse Brain Vessels at JoVE.com

Purification of Mouse Brain Vessels

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Research

JoVE Journal

Neuroscience

23.3K 次观看.  Institut National de la Santé et de la Recherche Médicale. We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes. 

视频: Purification of Mouse Brain Vessels

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

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 ...

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

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 ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

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 ...

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Purification of Mouse Brain Vessels

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