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>

<ol>
	<li>Nishida, N., Yano, H., Nishida, T., Kamura, T., Kojiro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+cancer.">Angiogenesis in cancer.</a> Vascular Health and Risk Management. 2 (3), 213-219 (2006).</li><li>Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+morphogenesis+and+the+formation+of+vascular+networks.">Vascular morphogenesis and the formation of vascular networks.</a> Developmental Cell. 6 (4), 479-482 (2004).</li><li>Magistretti, P. J., Zigmond, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brain energy metabolism in Fundamental neuroscience. , 389-413 (1999).</li><li>Weltzien, F. A., Andersson, E., Andersen, O., Shalchian-Tabrizi, K., Norberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain-pituitary-gonad+axis+in+male+teleosts,+with+special+emphasis+on+flatfish+(Pleuronectiformes).">The brain-pituitary-gonad axis in male teleosts, with special emphasis on flatfish (Pleuronectiformes).</a> Comparative Biochemistry and Physiology - Part A: Molecular &amp; Integrative Physiology. 137 (3), 447-477 (2004).</li><li>Ooi, G. T., Tawadros, N., Escalona, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+cell+lines+and+their+endocrine+applications.">Pituitary cell lines and their endocrine applications.</a> Molecular and Cellular Endocrinology. 228 (1-2), 1-21 (2004).</li><li>Knigge, K. M., Scott, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+median+eminence.">Structure and function of the median eminence.</a> American Journal of Anatomy. 129 (2), 223-243 (1970).</li><li>Ball, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+control+of+the+pars+distalis+in+fishes,+amphibians,+and+reptiles.">Hypothalamic control of the pars distalis in fishes, amphibians, and reptiles.</a> General Comparative Endocrinology. 44 (2), 135-170 (1981).</li><li>Knowles, F., Vollrath, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+contacts+between+neurosecretory+fibres+and+pituicytes+in+the+pituitary+of+the+eel.">Synaptic contacts between neurosecretory fibres and pituicytes in the pituitary of the eel.</a> Nature. 206 (4989), 1168 (1965).</li><li>Knowles, F., Vollrath, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosecretory+innervation+of+the+pituitary+of+the+eels+Anguilla+and+Conger+I.+The+structure+and+ultrastructure+of+the+neuro-intermediate+lobe+under+normal+and+experimental+conditions.">Neurosecretory innervation of the pituitary of the eels Anguilla and Conger I. The structure and ultrastructure of the neuro-intermediate lobe under normal and experimental conditions.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 250 (768), 311-327 (1966).</li><li>Golan, M., Zelinger, E., Zohar, Y., Levavi-Sivan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+GnRH-Gonadotrope-Vasculature+Reveals+a+Dual+Mode+of+Gonadotropin+Regulation+in+Fish.">Architecture of GnRH-Gonadotrope-Vasculature Reveals a Dual Mode of Gonadotropin Regulation in Fish.</a> Endocrinology. 156 (11), 4163-4173 (2015).</li><li>Isogai, S., Horiguchi, M., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+anatomy+of+the+developing+zebrafish:+an+atlas+of+embryonic+and+early+larval+development.">The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development.</a> Developmental Biology. 230 (2), 278-301 (2001).</li><li>Cha, Y. R., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+experimental+analysis+of+blood+vessel+formation+using+transgenic+zebrafish.">Visualization and experimental analysis of blood vessel formation using transgenic zebrafish.</a> Birth Defects Research Part C: Embryo Today. 81 (4), 286-296 (2007).</li><li>Kamei, M., Isogai, S., Pan, W., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+blood+vessels+in+the+zebrafish.">Imaging blood vessels in the zebrafish.</a> Methods Cell Biology. 100, 27-54 (2010).</li><li>Wu, E. S., Jacobson, K., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+Diffusion+in+Phospholipid+Multibilayers+Measured+by+Fluorescence+Recovery+after+Photobleaching.">Lateral Diffusion in Phospholipid Multibilayers Measured by Fluorescence Recovery after Photobleaching.</a> Biochemistry. 16 (17), 3936-3941 (1977).</li><li>Schlessinger, J., Axelrod, D., Koppel, D. E., Webb, W. W., Elson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+Transport+of+a+Lipid+Probe+and+Labeled+Proteins+on+a+Cell-Membrane.">Lateral Transport of a Lipid Probe and Labeled Proteins on a Cell-Membrane.</a> Science. 195 (4275), 307-309 (1977).</li><li>Johnson, M., Edidin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+Diffusion+in+Plasma-Membrane+of+Mouse+Egg+Is+Restricted+after+Fertilization.">Lateral Diffusion in Plasma-Membrane of Mouse Egg Is Restricted after Fertilization.</a> Nature. 272 (5652), 448-450 (1978).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbocyanine+Dye+Orientation+in+Red-Cell+Membrane+Studied+by+Microscopic+Fluorescence+Polarization.">Carbocyanine Dye Orientation in Red-Cell Membrane Studied by Microscopic Fluorescence Polarization.</a> Biophysical Journal. 26 (3), 557-573 (1979).</li><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Carbocyanine+Dyes+Allow+Living+Neurons+of+Identified+Origin+to+Be+Studied+in+Long-Term+Cultures.">Fluorescent Carbocyanine Dyes Allow Living Neurons of Identified Origin to Be Studied in Long-Term Cultures.</a> Journal of Cell Biology. 103 (1), 171-187 (1986).</li><li>Godement, P., Vanselow, J., Thanos, S., Bonhoeffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+in+developing+visual+systems+with+a+new+method+of+staining+neurones+and+their+processes+in+fixed+tissue.">A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue.</a> Development. 101 (4), 697-713 (1987).</li><li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+Fixation.">Formaldehyde Fixation.</a> Journal of Histochemistry &amp; Cytochemistry. 33 (8), 845-853 (1985).</li><li>Puchtler, H., Meloan, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Chemistry+of+Formaldehyde+Fixation+and+Its+Effects+on+Immunohistochemical+Reactions.">On the Chemistry of Formaldehyde Fixation and Its Effects on Immunohistochemical Reactions.</a> Histochemistry. 82 (3), 201-204 (1985).</li><li>Hoetelmans, R. W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acetone,+methanol,+or+paraformaldehyde+on+cellular+structure,+visualized+by+reflection+contrast+microscopy+and+transmission+and+scanning+electron+microscopy.">Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy.</a> Applied Immunohistochemistry &amp; Molecular Morphology. 9 (4), 346-351 (2001).</li><li>Hobro, A. J., Smith, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+fixation+methods:+Spatial+and+compositional+cellular+changes+observed+by+Raman+imaging.">An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging.</a> Vibrational Spectroscopy. 91, 31-45 (2017).</li><li>Li, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+labeling+and+visualization+of+blood+vessels+with+lipophilic+carbocyanine+dye+DiI.">Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI.</a> Nature Protocols. 3 (11), 1703-1708 (2008).</li><li>Wittbrodt, J., Shima, A., Schartl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medaka--a+model+organism+from+the+far+East.">Medaka--a model organism from the far East.</a> Nature Review Genetic. 3 (1), 53-64 (2002).</li><li>Fontaine, R., Hodne, K., Weltzien, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healthy+Brain-pituitary+Slices+for+Electrophysiological+Investigations+of+Pituitary+Cells+in+Teleost+Fish.">Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish.</a> Journal of Visual Experiments. (138), 57790 (2018).</li><li>Ager-Wick, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+a+High-quality+Primary+Cell+Culture+from+Fish+Pituitaries.">Preparation of a High-quality Primary Cell Culture from Fish Pituitaries.</a> Journal of Visual Experiments. (138), 58159 (2018).</li><li>Hildahl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+tracing+of+luteinizing+hormone+beta-subunit+gene+expression+using+green+fluorescent+protein+transgenic+medaka+(Oryzias+latipes)+reveals+a+putative+novel+developmental+function.">Developmental tracing of luteinizing hormone beta-subunit gene expression using green fluorescent protein transgenic medaka (Oryzias latipes) reveals a putative novel developmental function.</a> Developmental Dynamics. 241 (11), 1665-1677 (2012).</li><li>Schmid, B., Schindelin, J., Cardona, A., Longair, M., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-level+3D+visualization+API+for+Java+and+ImageJ.">A high-level 3D visualization API for Java and ImageJ.</a> BMC Bioinformatics. 11, 274 (2010).</li><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dil+and+diO:+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing.">Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends Neurosciences. 12 (9), 333-335 (1989).</li><li>Fontaine, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+Neurons+Controlling+Anterior+Pituitary+Functions:+Anatomy+and+Ontogenesis+in+Zebrafish.">Dopaminergic Neurons Controlling Anterior Pituitary Functions: Anatomy and Ontogenesis in Zebrafish.</a> Endocrinology. 156 (8), 2934-2948 (2015).</li></ol>

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 ...

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 border="0" cellpadding="0" cellspacing="0" style="width:877px;" width="877">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">16% paraformaldehyde</td>
			<td style="width:171px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">RT 15711</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">5 mL Syringe PP/PE without needle</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:123px;">Z116866-100EA</td>
			<td style="width:152px;">syringes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:432px;">BD Precisionglide syringe needles</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:123px;">Z118044-100EA</td>
			<td style="width:152px;">needles 18G (1.20*40)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">borosilicate glass 10cm OD1.2mm</td>
			<td style="width:171px;">sutter instrument</td>
			<td style="width:123px;">BF120-94-10</td>
			<td style="width:152px;">glass pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">DiI (1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate)</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:123px;">D-282</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:432px;">LDPE tube O.D 1.7mm and I.D 1.1mm</td>
			<td style="width:171px;">Portex</td>
			<td style="width:123px;">800/110/340/100</td>
			<td style="width:152px;">canula</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Phosphate Buffer Saline (PBS) solution</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:123px;">D8537-6X500ML</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">pipette puller</td>
			<td style="width:171px;">Narishige</td>
			<td style="width:123px;">PC-10</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">plastic petri dishes</td>
			<td style="width:171px;">VWR</td>
			<td style="width:123px;">391-0442</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Super glue gel</td>
			<td style="width:171px;">loctite</td>
			<td style="width:123px;">c4356</td>
			<td style="width:152px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">tricaine (ms-222)</td>
			<td style="width:171px;">sigma</td>
			<td style="width:123px;">E10521-50G</td>
			<td style="width:152px;"></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 ...

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

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

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

A Novel 3D Printed Bioreactor for Ex Vivo Culture of Blood Vessels in Perfusion

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Author Spotlight: EasyFlow - An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...

Enhancing CVD Research with Modified Langendorff Perfusion

Modified Langendorff Perfusion for Extended Perfusion Times of Rodent Cardiac Grafts

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and scientific advancement. As a result, innovation, improvement and/or repurposing of the available research toolset can provide improved testbeds for research advancement. Langendorff perfusion is an extremely valuable research technique for the field of CVD research that can be modified to accommodate a wide array of experimental needs. This tailoring can be achieved by personalizing a large number of perfusion parameters, including perfusion pressure, flow, perfusate, temperature, etc. This protocol demonstrates the versatility of Langendorff perfusion and the feasibility of achieving longer perfusion times (4 h) without graft function loss by utilizing lower perfusion pressures (30-35 mmHg). Achieving extended perfusion times without graft damage and/or function loss caused by the technique itself has the potential to eliminate confounding elements from experimental results. In effect, in scientific circumstances where longer perfusion times are relevant to the experimental needs (i.e., drug treatments, immunological response analysis, gene editing, graft preservation, etc.), lower perfusion pressures can be key for scientific success.

Author Spotlight: Advancing Cardiovascular Research &#8212; Tailored Langendorff Perfusion Techniques for Improved Experimental Outcomes

This article demonstrates the feasibility of achieving longer perfusion times (4 h) of murine cardiac grafts without function loss by employing lower (30-35 mmHg) than physiological (60-80 mmHg) perfusion pressures during Langendorff.

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and ...