We thank Dr Shinji Kanda for demonstration of cardiac perfusion with fixative solution in medaka, Ms Lourdes Carreon G Tan for help with medaka husbandry, and Mr Anthony Peltier for illustrations. This work was funded by NMBU and by the Research Council of Norway, grant number 248828 (Digital Life Norway program).

All animal handling was performed according to the recommendations for the care and welfare of research animals at the Norwegian University of Life Sciences, and under the supervision of authorized investigators.
1. Preparation of Instruments and Solutions
<ol>
	<li>Prepare DiI stock solution dissolving 5 mg of DiI crystal in 1.5 mL plastic tube with 1 mL of 96 % EtOH. Vortex for 30 s and keep covered using aluminum foil. 
	NOTE: The DiI stock solution can be conserved in the dark at -20 &#176;C for several months.</li>
	<li>Prepare the fish holder (Figure 1A) by cutting a piece of polystyrene to 5 cm length, 3 cm width, and 2 cm thickness, and gluing it to a 9 cm-diameter plastic dish. Make a 3 cm boat-shaped hole with a scalpel blade in the polystyrene.</li>
	<li>Prepare the perfusing system (Figure 2) by adding a 30-50 cm long plastic cannula at the extremity of the needle.</li>
	<li>Prepare 40 mL of fresh 4% paraformaldehyde solution (PFA) by diluting 10 mL of 16% PFA with 30 mL of phosphate buffered saline solution (PBS) in a 50 mL plastic tube.</li>
	<li>Prepare 10 mL of fixative/DiI solution by diluting 1 mL of DiI stock solution in 9 mL of freshly prepared 4% PFA in a 10 mL plastic tube. Keep in the dark until use. 
	NOTE: The DiI crystals that are not dissolved can be kept in the stock solution tube, and new ethanol can be added to prepare new DiI stock solution (see step 1.1).</li>
	<li>Prepare 50 mL of tricaine (MS-222) stock.
	<ol>
		<li>Dissolve 200 mg of Tricaine powder in 48 mL of H2O. Add 2 mL of 1 M Tris base (pH 9). Adjust to pH 7 with 1 M HCl and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Dilute 5 mL of Tricaine stock in 50 mL of clean water in a small glass.</li>
	<li>Prepare several 5 cm glass pipettes (Figure 2) by stretching a glass capillary with a pipette puller following the manufacturer&#39;s instructions.</li>
</ol>
2. Dissection and Perfusion
NOTE: PFA is a toxic volatile compound, therefore the dissection and perfusion should be performed in a hood or in a ventilated room, and the user must be wearing a gas mask.
<ol>
	<li>Prepare the dissection tools including small scissors, and one sharp and one strong forceps before dissection.</li>
	<li>Fill the syringe with the prepared solution of PFA/DiI by placing it in the 50 mL tube and drawing up. Then place the needle with the cannula at the extremity of the syringe (Figure 2).</li>
	<li>Fix the syringe to the bench using several pieces of tape placed in different directions, adjust the position of the microscope and the seat to obtain a good position for dissection and for pressing the syringe piston (Figure 1B). 
	NOTE: In this protocol, the elbow is used to press down the syringe piston, but other possibilities options may be used, e.g., assistance from another person.</li>
	<li>Euthanize the fish with an overdose of tricaine by placing the fish in the solution prepared in step 1.7. Wait 30 s and test the reflexes of the fish by grabbing its caudal fin with the forceps. The fish can be used when it has stopped responding to the stimuli.</li>
	<li>Place the fish in the fish holder with its abdomen facing up and pin one needle into the extremity of the head and another one above the tail to keep the fish in place.</li>
	<li>Open the anterior abdomen with the scissors by horizontally cutting the superficial layer of skin.</li>
	<li>Using forceps, remove the skin above the heart until a clear access to the ventricle and the bulbus arteriosus is provided (Figure 3).</li>
	<li>Add a glass pipette at the extremity of the capillary and break the end of the tip of the glass pipette. 
	NOTE: The hole of the broken tip should be big enough to let the liquid out, but still small enough to easily enter the tissue. The glass pipette can be reused if not broken or clogged.</li>
	<li>Bring the glass pipette close to the ventricle and add pressure to the syringe piston with the elbow to force the liquid out.</li>
	<li>Pin the heart ventricle with the glass pipette while adding pressure to the syringe.</li>
	<li>Directly after, perforate the sinus venosus with forceps to enable blood to leave the circulation. 
	NOTE: The heart ventricle becomes more fragile because of the fixative. It also becomes less red as the blood is diluted with the fixative/DiI solution.</li>
	<li>From the ventricle, adjust the angle of the glass pipette to find the entrance of the bulbus arteriosus. Bring the pipette opening inside the bulbus arteriosus as shown in Figure 2, and add more pressure to the syringe. 
	NOTE: The bulbus arteriosus is transparent and the tissue is elastic. When replacing blood with DiI/fixative, the glass pipette inside the heart should become more visible and by adding pressure, the size of the bulbus arteriosus should expand. This step is crucial to make sure that enough pressure has been used to replace all the blood with the fixative/DiI solution, and that all blood vessels have been reached. Often when successful, the PFA provokes muscle contractions and the fish will be moving.</li>
	<li>Continue adding pressure on the syringe for 30-60 s still keeping the glass pipette inside the bulbus arteriosus.</li>
	<li>Remove the glass pipette and the needles from the fish.</li>
	<li>Dissect the brain and the pituitary, and incubate tissues in fresh 4% PFA in PBS for 2 h in the dark at room temperature. 
	NOTE: Dissection of brain and pituitary previously has been described in detail in two different ways by Fontaine et al.26&#160;and Ager-Wick et al.27. Because of the fixation, the tissue becomes quite hard and the fragile link between the brain and the pituitary may be broken. After dissection, post fixation processing can also be performed overnight at 4 &#176;C.</li>
	<li>Rinse the tissue twice in PBS for 10 min before preparing for imaging. 
	NOTE: The tissue can then be mounted between slide and cover slip with spacers in between and mounting medium for confocal imaging (see Figure 4), or sectioned with a vibratome as described in detail in Fontaine et al.26&#160;before mounting between slide and cover slip for imaging with a stereomicroscope (see Figure 4).</li>
</ol>

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The authors have nothing to disclose

Cardiac perfusion with DiI previously has been used to label blood vessels in several model species24, including teleost fish13.
As DiI is directly delivered to the endothelial cell membrane by perfusion in the vasculature, it is possible to increase the signal-to-noise ratio by increasing the DiI concentration in the fixative solution. In addition, the fluorophore provides intense staining when excited with minimal bleaching allowing relatively long-lasting emission18,30.&#160;Also, the labeling can remain for several days and be used in combination with other labeling techniques that require mild treatments, such as in immuofluorescence (IF)31.
However, this technique has some limitations. After removal from the fixative solution, labeling and imaging of the tissues should be performed as quickly as possible because the intensity of the labeling will weaken, and the signal will diffuse with time (Figure 8). This process will be even faster when using detergents that increase porosity of the membrane. Also, because PFA is a toxic volatile compound, several precautions should be taken when performing this experiment. To minimize the hazardous aspects of of this protocol, one can perfuse a solution of DiI and PBS before dissecting the tissue of interest and fix it with the PFA solution right after perfusion. However, this can be performed only for small-sized tissues, as the penetration of PFA in larger tissue samples will be less efficient than during perfusion.
The success rate of this protocol is improved by training, so it is expected that researchers will need some time to get acquainted with the different steps of the technique. For instance, the perfusion of the solution in the bulbus arteriosus is a particularly critical step of the protocol and requires some training to achieve a good result. Also keeping the needle in the correct position for long enough during the perfusion is not trivial, and will require training by the manipulator.
Finally, this protocol is optimized for adult medaka but it can be used for other species with some adaptations. Different tissues of interest, or even different life stages of the animal within the same tissue will also require optimizations for the concentration of DiI and the time of perfusion as well as the material used (needle and syringe sizes for example).&#160;

Blood vessels play an essential part of the vertebrate body as they provide the necessary nutrients, oxygen and hormonal signals to all organs. Also, since the discovery of their involvement in cancer development1, they have received much attention in clinical research. Although a number of publications have investigated the mechanisms allowing blood vessel growth and morphogenesis, and a large number of genes important for their formation have been identified2, a lot remains to be understood regarding the interaction between cells or tissues and the circulating blood.
Visualization of blood vasculature in the brain and pituitary is important. Neurons in the brain require a high supply of oxygen and glucose3, and the pituitary contains up to eight important hormone-producing cell types that use the blood flow to receive signal from the brain and send their respective hormones to different peripheral organs4,5. While in mammals, the portal system at the base of the hypothalamus named the median eminence, links the brain and the pituitary6, such a clear blood bridge has not been described in teleost fish. Indeed, in teleosts, preoptico-hypothalamic neurons directly project their axons into the pars nervosa of the pituitary7 and mostly innervate the different endocrine cell types directly8,9. However, some of these neurons have their nerve endings located in the extravascular space, in close vicinity to blood capillaries10. Therefore, the difference between teleost fish and mammals is not so clear, and the relationship between the blood vasculature and the brain and pituitary cells requires greater investigation in teleost fish.
Zebrafish has, in many aspects, an anatomically and functionally comparable vascular system to other vertebrate species11. It has become a powerful vertebrate model for cardiovascular research mostly thanks to the development of several transgenic lines where components of the vascular system are labeled with fluorescent reporter proteins12. However, exact circulatory system anatomy may vary between species, or even between two individuals belonging to the same species. Therefore, visualization of blood vessels may be of high interest also in other teleost species for which transgenesis tools do not exist.
Several techniques have been described to label blood vessels in both mammals and teleosts. These include in situ hybridization for vasculature-specific genes, alkaline phosphatase staining, microangiography, and dye injections (for a review see13). Fluorescent lipophilic cationic indocarbocyanine dye (DiI) was first used to study membrane lipids lateral mobility as it is retained in the lipid bilayers and can migrate through it14,15,16. Indeed, a molecule of DiI is composed of two hydrocarbon chains and chromophores. While the hydrocarbon chains integrate in the lipid bilayer cell membrane of the cells in contact with it, the chromophores remain on its surface17. Once in the membrane, DiI molecules diffuse laterally within the lipid bilayer which helps to stain membrane structures that are not in direct contact with the DiI solution. Injecting a DiI solution through cardiac perfusion, will therefore label all endothelial cells in contact with the compound allowing direct labelling of the blood vessels. Today DiI is also used for other staining purposes, such as single molecule imaging, fate mapping, and neuronal tracing. Interestingly, several fluorophores exist (with different wavelengths of emission) allowing the combination with other fluorescent labels, and the incorporation as well as the lateral diffusion of DiI can occur in both live and fixed tissues18,19.
Formaldehyde, discovered by Ferdinand Blum in 1893, has been used widely to the present day as the preferred chemical for tissue fixation20,21. It shows broad specificity for most cellular targets and preserves the cellular structure22,23. It also preserves the fluorescent properties of most fluorophores, and thus can be used to fixate transgenic animals for which targeted cells express fluorescent reporter proteins.
In this manuscript, a previous protocol developed to label blood vessels in small experimental mammalian models24&#160;has been adapted to the use in fish. The entire procedure takes only a couple of hours to perform. It demonstrates how to perfuse a fixative solution of formaldehyde containing DiI in the fish heart in order to directly label all blood vessels in the brain and the pituitary of the model fish medaka. Medaka is a small freshwater fish native to Asia, primarily found in Japan. It is a research model organism with a suite of molecular and genetic tools available25. Therefore, identification of blood vessels in this species as well as in others will allow to improve our understanding on how the brain and pituitary cells interact with blood vasculature in whole tissue or thick tissue slices.

<table><tbody><tr><td>16% paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>RT 15711</td><td></td></tr><tr><td>5 mL Syringe PP/PE without needle</td><td>Sigma</td><td>Z116866-100EA</td><td>syringes</td></tr><tr><td>BD Precisionglide syringe needles</td><td>Sigma</td><td>Z118044-100EA</td><td>needles 18G (1.20*40)</td></tr><tr><td>Borosilicate glass 10 cm OD 1.2 mm</td><td>sutter instrument</td><td>BF120-94-10</td><td>glass pipette</td></tr><tr><td>DiI (1,1&amp;prime;-Dioctadecyl-3,3,3&amp;prime;,3&amp;prime;-tetramethylindocarbocyanine perchlorate)</td><td>Invitrogen</td><td>D-282</td><td></td></tr><tr><td>LDPE tube O.D 1.7 mm and I.D 1.1 mm</td><td>Portex</td><td>800/110/340/100</td><td>canula</td></tr><tr><td>Phosphate Buffer Saline (PBS) solution</td><td>Sigma</td><td>D8537-6X500ML</td><td></td></tr><tr><td>Pipette puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Plastic Petri dishes</td><td>VWR</td><td>391-0442</td><td></td></tr><tr><td>Super glue gel</td><td>loctite</td><td>c4356</td><td></td></tr><tr><td>Tricaine (ms-222)</td><td>sigma</td><td>E10521-50G</td><td></td></tr></tbody></table>

labeling of blood vessels in the teleost brain and pituitary using cardiac perfusion with a dii-fixative

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one of the first organs to start functioning during development. Mechanisms of blood vessel formation have become a subject of high scientific and clinical interest. In adults however, it is difficult to visualize the vasculature in most living animals due to their localization deep within other tissues. Nevertheless, visualization of blood vessels remains important for several studies such as endocrinology and neurobiology. While several transgenic lines have been developed in zebrafish, with blood vessels directly visualized through expression of fluorescent proteins, no such tools exist for other teleost species. Using medaka (Oryzias latipes) as a model, the current protocol presents a quick and direct technique to label blood vessels in brain and pituitary by perfusing through the heart with fixative containing DiI. This protocol allows improvement of our understanding on how brain and pituitary cells interact with blood vasculature in whole tissue or thick tissue slices.

This protocol demonstrates a step by step procedure to label blood vessels in the medaka brain and pituitary, and at the same time fix the tissue. After labelling by cardiac injection of a fixative solution containing DiI into the heart, blood vessels can be observed on slices using a fluorescent stereomicroscope (Figure 4) or on whole tissue using a confocal microscope (Figure 5). Either on the thick tissue slice or on the whole tissue, the architecture of the blood vasculature can be observed in three dimensions. Tissue slices can be labeled for specific targeted proteins using standard immunofluorescence protocols after the end of the fixation, and on transgenic lines where cells of interest express fluorescent reporter proteins (Figure 6). This allows investigations on interactions between blood vessels and other specific cell types. Here for example, the use of a transgenic line of medaka where green fluorescent protein (GFP)-producing cells revealed the location of pituitary Lh cells28. One can observe that these cells send extensions towards the blood vessels. Some blood vessels may not be labeled properly if perfusion is sub-optimal. This can be the case, for example, if the solution is injected in the heart ventricle instead of the bulbus arteriosus, or if using too low pressure and/or for too short a period on the syringe piston (Figure 7). Finally, by imaging the same tissue with the same imaging parameters, the intensity of the labeling was shown to decrease after four days, with the signal more spread out (Figure 8).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59768/59768fig1.jpg" /> 
Figure 1: Images of the holding plate for perfusion of the fish (A) and the injection setup (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59768/59768fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59768/59768fig2.jpg" /> 
Figure 2: Schema of the medaka fish heart and perfusion system shown with the idel location of the glass needle in the heart for successful perfusion. Arrow head show where to perforate the heart to allow the blood to leave circulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59768/59768fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59768/59768fig3.jpg" /> 
Figure 3: Image of the ventral side of the fish opened, with the heart exposed for perfusion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59768/59768fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59768/59768fig4.jpg" /> 
Figure 4: Image of the blood vasculature in a slice of brain and pituitary tissue from medaka. An adult medaka was perfused with a fixative solution containing DiI. Brain and pituitary were dissected and fixed overnight. Tissues were mounted in 3% agarose and sectioned with a vibratome before imaging with a fluorescence microscope. All blood vessels labeled with DiI are seen through the whole section showing the vasculature in the brain and the pituitary. OT = optic tectum; Tel = telencephalon; Hyp = hypothalamus; Pit = pituitary; Cb = cerebellum; Hind = hindbrain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59768/59768fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59768/59768fig5.jpg" /> 
Figure 5: 3D rendering of a confocal z-stack from the blood vasculature in medaka pituitary. An adult medaka was perfused with a fixative solution containing DiI. Brain-pituitary were dissected and fixed overnight. The pituitary was dissected from the brain and mounted between a slide and coverslip with spacers ib between, before imaging with a confocal microscope. Z-stack was recorded, and a 3D rendering was made using Fiji software and the 3D viewer plugin29. The entire pituitary blood vasculature could be observed in 3D. RPD = rostral pars distalis; PPD = Proximal pars distalis; PI = pars intermedia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59768/59768fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59768/59768fig6.jpg" /> 
Figure 6: Z projection of confocal z-stack from a tissue slice of transgenic medaka where Lh&#946; promoter controls expression of green fluorescent reporter protein. An adult tg(lhb:hrGfpII) medaka was perfused with a fixative solution containing DiI. Brain-pituitary were dissected and fixed overnight. Tissues were mounted in 3% agarose and sectioned with a vibratome. Some hormone producing cells, Lh cells in this case, sending extensions (arrow heads) towards the blood vessels can be observed, probably to sense signals from the blood and/or release their hormones into the circulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59768/59768fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59768/59768fig7.jpg" /> 
Figure 7: Image of blood vasculature in a slice of brain and pituitary tissue from poorly perfused medaka. An adult medaka was poorly perfused with a fixative solution containing DiI by injecting the solution in the ventricle only. Brain-pituitary were dissected and fixed overnight. Tissues were mounted in 3% agarose and sectioned with a vibratome before imaging with a confocal microscope. The blood vessels were poorly labeled with DiI and some of them were not labeled at all. OT = optic tectum; Tel =, telencephalon; Hyp = hypothalamus; Pit = pituitary; Cb = cerebellum; Hind = hindbrain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59768/59768fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59768/59768fig8.jpg" /> 
Figure 8: Time lapse imaging of labeled blood vasculature in medaka brain-pituitary tissue section. An adult medaka was perfused with a fixative solution containing DiI. Brain-pituitary were dissected and fixed overnight. Tissues were mounted in 3% agarose and sectioned with a vibratome before imaging with a stereomicroscope directly after mounting (A), and 4 days after mounting (B) with the same imaging parameters. Note that the labeling has decreased and spread out with time. OT = optic tectum; Tel = telencephalon; Hyp = hypothalamus; Pit = pituitary; Cb = cerebellum; Hind = hindbrain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59768/59768fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative at JoVE.com

Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

The article describes a quick protocol for labeling blood vessels in a teleost fish by cardiac perfusion of DiI diluted in fixative, using medaka (Oryzias latipes) as a model and focusing on brain and pituitary tissue.

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one ...

labeling-blood-vessels-teleost-brain-pituitary-using-cardiac

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.4K Views.  Norwegian University of Life Sciences. The article describes a quick protocol for labeling blood vessels in a teleost fish by cardiac perfusion of DiI diluted in fixative, using medaka (Oryzias latipes) as a model and focusing on brain and pituitary tissue.

Video: Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Neuroscience

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Immunology and Infection

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.

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 ...

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The cerebral vasculature dynamically responds to various pathophysiological states and it exhibits considerable differences between strains and under conditions of genetic manipulations. Essentially, a reliable technique for intracranial vessel staining is essential in order to study the pathogenesis of ischemic stroke. Until recently, a set of different techniques has been employed to visualize the cerebral vasculature including injection of low viscosity resin, araldite F, gelatin mixed with various dyes1 (i.e. carmine red, India ink) or latex with2 or without3 carbon black. Perfusion of white latex compound through the ascending aorta has been first reported by Coyle and Jokelainen3. Maeda et al.2 have modified the protocol by adding carbon black ink to the latex compound for improved contrast visualization of the vessels after saline perfusion of the brain. However, inefficient perfusion and inadequate filling of the vessels are frequently experienced due to high viscosity of the latex compound4. Therefore, we have described a simple and cost-effective technique using a mixture of two commercially available carbon black inks (CB1 and CB2) to visualize the cerebral vasculature in a reproducible manner5. We have shown that perfusion with CB1+CB2 in mice results in staining of significantly smaller cerebral vessels at a higher density in comparison to latex perfusion5. Here, we describe our protocol to identify the anastomotic points between the anterior (ACA) and middle cerebral arteries (MCA) to study vessel variations in mice with different genetic backgrounds. Finally, we demonstrate the feasibility of our technique in a transient focal cerebral ischemia model in mice by combining CB1+CB2-mediated vessel staining with TTC staining in various degrees of ischemic injuries.

Analysis of rodent cerebrovascular anatomy plays an important role in experimental stroke research. In this context, intravascular perfusion with colored latex has been considered as a standard tool for several years. However, this technique implies distinct technical limitations, which undermine its reproducibility. Here, we describe a simple method to visualize cerebral vessels in a reproducible manner. Injection of a mixture of two commercially available carbon black inks through the left myocardial ventricle results in adequate filling of cerebral vessels with high contrast visualization. We have successfully applied this technique to identify anastomotic points between cerebral vascular territories of mice with different genetic backgrounds. We finally give evidence that this novel and simple method for vessel staining can be combined with triphenyltetrazolium chloride (TTC) staining - a widely used tool to observe and analyze infarct volumes in mice.

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

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.

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 ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Visualization of Brain Pericyte Contractility Post-Subarachnoid Hemorrhage

Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility adjustments. Conventionally, their contractility is gauged by observing morphological shifts and nearby capillary diameter changes under specific circumstances. Yet, post-tissue fixation, evaluating vitality and ensuing pericyte contractility of imaged brain pericytes becomes compromised. Similarly, genetically labeling brain pericytes falls short in distinguishing between viable and non-viable pericytes, particularly in neurologic conditions like subarachnoid hemorrhage (SAH), where our preliminary investigation validates brain pericyte demise. A reliable protocol has been devised to surmount these constraints, enabling simultaneous fluorescent tagging of both functional and non-functional brain pericytes in brain sections. This labeling method allows high-resolution confocal microscope visualization, concurrently marking the brain slice microvasculature. This innovative protocol offers a means to appraise brain pericyte contractility, its impact on capillary diameter, and pericyte structure. Investigating brain pericyte contractility within the SAH context yields insightful comprehension of its effects on cerebral microcirculation.

Author Spotlight: Imaging Pericytes Post-Subarachnoid Hemorrhaging in Rodents 

The preliminary inquiry confirms that subarachnoid hemorrhage (SAH) causes brain pericyte demise. Evaluating pericyte contractility post-SAH requires differentiation between viable and non-viable brain pericytes. Hence, a procedure has been developed to label viable and non-viable brain pericytes concurrently in brain sections, facilitating observation using a high-resolution confocal microscope.

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

Summary

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Chapters in this video

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

A Method for Labeling Vasculature in Embryonic Mice

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Purification of Mouse Brain Vessels

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Free-floating Immunostaining of Mouse Brains

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum