Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

Summary

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Abstract

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

Introduction

The zebrafish is currently one of the most important vertebrate models to study cellular and developmental processes in vivo. This is largely due to the zebrafish's optical transparency and amenability to genetics, which makes it a powerful model for applying optical techniques involving genetically encoded photoresponsive protein technologies¹. Specific to the study of heart development, zebrafish receive sufficient oxygen via diffusion such that even mutants without heartbeat can survive through the first week of development, permitting analyses on the effects of developmental genes and perturbed blood flow on heart morphogenesis not possible in most vertebrates².

The zebrafish heart tube is formed by 24 hours post fertilization (hpf). Shortly after its formation, the heart tube starts actively beating. By 36 hpf, a clear constriction separates the atrium from the ventricle. This region of constriction is called the atrioventricular canal (AVC), and cells in this region change from a squamous morphology to a cuboidal morphology³. Zebrafish atrioventricular valve morphogenesis starts around 48 h post fertilization. By 5 days post fertilization, two valve leaflets extend into the AVC and prevent the back flow of blood from the ventricle to the atrium during the cardiac cycle⁴. Tracking cells during AVC and valve formation is challenging as the rapid beating of the heart makes it difficult to follow cells via traditional time-lapse microscopy^5,6. This protocol, adapted from Steed et al., 2016⁷, describes a method that uses the Tg(fli1a:gal4FF^ubs, UAS:kaede)⁸ zebrafish transgenic line, in which the photoconvertible protein Kaede is expressed in endothelial cells, including the endocardium. The drug 2,3-butanedione-2-monoxime (BDM) is used to temporarily stop the heart beating, allowing accurate photoconversion of EdCs between 36 and 55 hpf, and high-resolution imaging of photoconverted EdCs at specific developmental time points. It has previously been shown that EdCs photoconverted using this method can remain distinguishable from their unconverted neighbors for five days or more after the time of photoconversion⁷. This protocol also details a method used for image analysis of photoconverted EdCs in embryos younger than 48 hpf, which has successfully been used to follow tissue movements during AVC development (Boselli et al., in press)⁹. We hope that readers would find this protocol useful for studying AVC and valve development in normal embryos, and in mutants, morphants, or drug-treated embryos. For a more general protocol relating to cell tracking using photoconvertible proteins during zebrafish development, please view the article by Lombardo et al., 2012¹⁰.

Protocol

1. Preparing molds and mounting agarose

1. Create a mold with the dimensions shown in Figure 1 using either a 3D printer or traditional mechanical workshop tools.
2. Pipette about 1.5 mL of melted 1 % agarose into a 35-mm plastic mounting dish. Place the plastic mold in the liquid agarose, taking care to avoid trapping air between the mold and the agarose. Place the dish at 4 °C, wait till the agarose hardens (this takes about 5 min), then remove the plastic mold.
3. To store the mounting dish for later use, cover the dish with its lid, then wrap both the dish and the lid tightly with parafilm. The mounting dish can then be stored lid side facing down for up to 5 days at 4 °C.
4. Prepare in advance 1 mL aliquots of 0.7 % low melting point agarose in 1.5 mL Eppendorf tubes to use on the day of mounting.
   NOTE: The agarose wells of the mounting dish will deform over time as water evaporates if parafilm is not wrapped around the dishes tightly.

2. Obtaining Embryos for the Photoconversion

1. Incross the Tg(fli1a:gal4FF^ubs, UAS:kaede) line and grow approximately 200 embryos in embryo medium in the dark at 28.5 °C. For more details on how to cross zebrafish lines, please refer to the JoVE Science Education Database¹¹.
2. After 5 h but before 24 h, treat embryos with 1-phenyl-2-thiourea (PTU) to prevent pigment formation (0.003 % PTU in embryo medium).
3. About 1 h before the start of photoconversion, screen embryos under a fluorescent stereoscope and select 5 healthy embryos that appear to express the brightest green fluorescence. The use of low-intensity light when visualizing embryos is recommended to avoid exposure of the embryos to ambient light in order to avoid photoconverting Kaede in non-specific cells.

3. Embedding

1. Prepare 10 mL of mounting media: 0.02 % tricaine and 50 mM BDM in embryo medium.
2. Add 2 mL of mounting medium to the mounting dish and allow 15-30 min for the solution to diffuse into the agarose.
3. In the meantime, melt a 1 mL aliquot of 0.7 % low melting point (LMP) agarose by placing the tube in a 70 °C heat block for about 5 min.
   1. Wait for the LMP agarose to cool slightly, then add 25 µL of 8 mg/mL tricaine solution and 40 µL of 1M BDM solution to the tube of low melting point agarose. Mix by pipetting up and down, then transfer the tube to a 38 °C heat block to keep the LMP agarose in its liquid state.
4. In a Petri dish with embryo medium, carefully dechorionate the pre-selected embryos under the stereomicroscope using forceps, taking care not to damage the embryos.
5. Transfer the embryos to a separate dish containing 2 mL of mounting media.
   1. When the hearts of the embryos are stopped (takes about 5-10 min), transfer the embryos to the mounting dish and arrange embryos in the wells using forceps so that they lie at a 25 degree angle, tails pointing towards the deeper part of the well, yolk facing up.
   2. When embryos are roughly in place, remove the media, embed the embryos in ~200 µL of 0.7 % LMP agarose containing tricaine and BDM, and readjust the embryos' positions, tilting the embryos to the left or right as necessary.
6. Wait until the LMP agarose sets (takes about 5 min), then add mounting medium to the dish. The embryos are now ready to be photoconverted.
   NOTE: The tricaine and the BDM are used to anesthetize the embryos and to stop the embryos' heart, respectively. Mounting media can be prepared the day before imaging, but make sure to protect the media from light.
   NOTE: The correct positioning of the embryos is important to allow a clear laser path to the cells one wishes to photoconvert. Position the head of the embryo close to the start of the well so that laser light does not have to travel far to reach the embryo, and so that the embryos can be easily unmounted after photoconversion.

4. Photoconversion

1. On an upright confocal microscope (like Leica SP8) equipped with a 405 nm, 488 nm and 561 nm laser source, locate the embryo's heart using transmitted white light (brightfield) and an objective lens (like Leica HCX IRAPO L, 25 ×, N.A. 0.95 objective). Check for red Kaede fluorescence using the 561 nm laser (i.e., DPSS 561 laser, at ~5 % intensity, detector set at 589-728 nm). Then, visualize the endocardium by using the 488 nm laser (i.e., 30 mW multiline Argon laser at ~5 % intensity, detector set at 502-556 nm).
2. Enter "FRAP" mode on the microscope acquisition software.Select ~1 % laser power for both 488 nm and 561 nm lasers.
3. Focus on the plane of interest, select a region of interest (ROI), then photoconvert cells using the 405 nm diode laser at 25 % laser power, scanning over the ROI three times. Should insufficient Kaede be photoconverted in the ROI, scan the 405 nm laser over the area three times again. Repeat this step for all ROIs.
4. Return to the "TCS SP8" mode on the software and acquire a z-stack, using the 561 nm and 488 nm lasers sequentially. Make sure to select the 'bidirectional' option to increase imaging speed.
   NOTE: How long it takes to photoconvert and image each embryo varies. Heart beat is known to be important for normal heart valve development, so avoid keeping the heart stopped for more than 30 min, even if it means you do not have time to photoconvert all 5 embryos.
   NOTE: During the photoconversion process, use PMT detectors, while after photoconversion, it is recommended to use hybrid detectors set to 'counting' mode. This is because the hybrid detectors are more sensitive and are capable of producing better quality images, but are easily damaged by overexposure.

5. Unmounting photoconverted embryos

1. After photoconversion, use a glass pipette to press down slightly just above the head of the embryo to break the LMP agarose, then gently suck up the embryo.
   1. Eject the embryo from the glass pipette into a 35-mm Petri dish containing embryo medium with PTU (to wash away BDM containing medium).
   2. Transfer the embryo to a well in a 6-well plate containing embryo medium with PTU. During this step, make sure to keep note of which embryo goes into which well, as this is essential to correlate the position of photoconverted cells at later developmental stages.
2. Now that the embryos are removed from the BDM containing medium, their hearts should start beating again in about 5 min. Return embryos to the dark at 28.5 °C to allow embryos to continue to develop normally.

6. Imaging photoconverted cells at later embryonic stages

1. At the desired stage, stop the heart and re-embed the embryos like under Step 3 of this protocol, with the important difference that embryos must be treated with BDM in separate, marked dishes to keep track of embryos.
2. For embryos up to 62 hpf, acquire z-stacks of the endocardium using 561 nm and 488 nm for red and green fluorescent signals, respectively. For embryos at stages older than 62 hpf, use a multiphoton laser set to 940 nm to image green Kaede instead of the 488 nm laser.

7. Image analysis for embryos under 48 hpf

Note: Tissue movements of the AVC can be difficult to analyze due to its complex three-dimensional structure. To quantify the length of a photoconverted region in the AVC between two non-photoconverted regions or the length of a non-photoconverted region in the AVC between two photoconverted regions, it can be desirable to map the three-dimensional structure onto a two-dimensional map. Images of photoconverted embryos obtained prior to 48 hpf can be analyzed using the following method.

1. Import the acquired images in Matlab or any similar software.
2. Delimitate manually the ROI, i.e. the endocardium, and apply a mask to remove the signal outside this region.
3. Apply an intensity threshold on the image to identify the points on the endocardium and plot them in three-dimensions. Isolate the points corresponding to the narrowest and most straight part of the AVC, by erasing manually the points of no interest on the atrium and the ventricle using the erase command in the software.
4. Use a cylinder to fit the points representing the AVC so obtained.
5. Define a reference system to compare several samples. For example, adopt the center of mass of the AVC points as the origin of the reference system and the axis of the fitted cylinder as the z-axis.
   1. Make sure that positive z-positions correspond always to the same side of the heart (ventricular or atrial).
   2. Project the AVC points on the x-y plane and use an ellipse to fit them.
   3. Rotate the endocardial points around the z-axis so that the major axis of the ellipse and the y-axis of the reference system are parallel and positive y-values correspond to the internal side of the AVC with respect to the embryo.
6. To segment the AVC, cut the three-dimensional dataset with some planes obtained by rotating the half-plane {y=0, x>0} in the z-direction.
   1. Project the intensity of the neighboring pixels on these planes and define the endothelium in a consistent manner. For example, draw manually a line from the atrial to the ventricular side of the AVC at half thickness of the endothelium.
   2. Interpolate the points of the lines representing the endothelium with a three-dimensional spline using the spline toolbox to represent the AVC with a surface.
      NOTE: The number of the cutting planes determines both the accuracy of the segmentation and the time consumption of this step. Usually, 8 planes are enough to achieve good results.
7. Define the azimuthal position of each point on the AVC surface using cylindrical coordinates.
   1. At each azimuthal position define a parametric curve along the surface of the AVC by calculating the arc length on the surface between consecutive points.
   2. For each parametric curve, define the point on the curve closest to the center of the AVC to zero. The points of the AVC are thus fully described by their azimuthal position and their position on the parametric curve.
8. Unfold the AVC into a two-dimensional image by using the parametric position of the points.
9. Find the edges of the region to quantify as the edges of the binary image thresholding the ratio between the intensities of the photoconverted and the non-photoconverted channels. Manually correct the result if necessary.
10. Calculate the arc length between the edges to compute the length of the tissue as a function of the azimuthal position.

Representative Results

An example of an embryo photoconverted at 48 hpf and imaged again at 80 hpf is shown in Figure 2, Movies 1 and 2. Exposing Kaede to 405 nm light irreversibly converts from the protein from its fluorescent green form to fluorescent red form, enabling the behavior of cells labeled with either the green or their red form to be followed with respect to their differentially colored neighbors during valve formation. It can be seen that the few photoconverted cells of the 48 hpf AVC later proliferated and later resided predominantly on one side of the valve leaflet.

An example of the results that can be achieved by applying the image analysis method described on a 36 hpf embryo after photoconverting EdCs in the atrium and in the ventricle is shown in Figure 3. The segmentation of the AVC allows the parametrization of the three-dimensional structure that is then projected in two-dimensions for easier visualization and analysis. The method allows one to quantify the distance between the two photoconverted regions. By repeating this process at different developmental times it is possible to study the migration of cells toward the AVC (Boselli et al., in press)⁹.

Figure 1: Mold used to create agarose wells. A) Front view. B) Back view. C-D) Side views. E-F) Oblique views. Dimensions are shown in millimeters. The .STL file for the mold template is provided in the Supplementary Materials. Please click here to view a larger version of this figure.

Figure 2: Representative results of a Tg(fli1a:gal4FF^ubs, UAS:kaede) photoconverted embryonic heart. A) The embryonic heart just after photoconversion at 48 hpf. Cytoplasmic Kaede within several cells of the superior side of the AVC has been converted from its green form to its red form. B) The same embryo at 80 hpf. It can be seen that the previously photoconverted cells have proliferated and have come to reside in the inner side of the superior atrioventricular valve. Scale bars: 20 µm. Please click here to view a larger version of this figure.

Figure 3: Representative results of the image analysis process on a 36 hpf embryo. A) The result of the three-dimensional segmentation of the AVC in a reference system that is common among the embryos; the signal from the photoconverted (magenta) and non-photoconverted (green) Kaede is projected onto its surface and marks the photoconverted and non-photoconverted regions. B) The AVC is unfolded according to the parametric position of its point and the signal is projected in two-dimensions for easier visualization. The edges of the non-photoconverted region are labeled in white. C) The length of the non-photoconverted region is plotted as a function of the angular position. Please click here to view a larger version of this figure.

Movie 1: The embryonic heart just after photoconversion at 48 hpf. Related to Figure 2A. The movie goes through the acquired z stack of the 48 hpf embryo shown in Figure 2A z slice by z slice. Only one z plane was chosen during the photoconversion process, but it can be seen here that cells at various depths have been photoconverted. Z slices are spaced 2 µm apart. Right click to download this movie.

Movie 2: The photoconverted embryonic heart at 80 hpf. Related to Figure 2B. The movie goes through the acquired z stack of the 80 hpf embryo shown in Figure 2B z slice by z slice. Z slices are spaced 2 µm apart. Right click to download this movie.

Supplementary file: Related to Figure 1. The .STL file for the mold template. Please click here to download this file.

Discussion

Timing of photoconversion: Although Kaede remains brightly expressed in EdCs even at 96 hpf, it should be noted that as the embryo grows, laser light diffuses more before it reaches the AVC, making confined photoconversion of Kaede more difficult. At embryonic stages later than 55 hpf, the ballooning of the ventricle and the atrium also means that the violet laser beam used for photoconversion cannot reach AVC cells without first passing through either the atrium or ventricle. This means that beyond 55 hpf, in order to photoconvert EdCs of the AVC, one must also photoconvert additional EdCs in the atrium and ventricle.

BDM and tricaine: Accurate photoconversion of EdCs requires temporarily stopping the heart from beating. BDM (2,3-butanedione monoxime) is an electro-contractile uncoupler that is well known to suppress cardiac muscle contraction¹². We do not observe differences between the morphology of embryos that have been subject to this protocol, and embryos that have been growing normally and have not had their hearts stopped for imaging. However, the risk remains that the drug treatment is altering normal development¹³. Concentrated tricaine can also be used to temporarily stop the heart¹⁴. We find that we can effectively stop the heart by placing embryos in embryo medium with 0.32 % tricaine for 1 min, and maintain the heart in its stopped state by transferring the embryos to embryo medium with 0.16 % tricaine. Similar to the BDM treatment, the heart can be stopped this way for 30 min and heart beat can be seen to return within 5 min after returning embryos into normal embryo medium. Higher concentrations of tricaine should be avoided; low concentrations of tricaine for prolonged periods (h/days) has been shown to affect normal embryonic development^15,16. Tricaine solution used for stopping the heart is best prepared fresh. If frozen aliquots are used, make sure to keep the solution well protected from light and to defrost the tricaine without heating the solution above 30 °C. Finally, blebbistatin has been reported to stop the heart without severe side effects¹³. We have not tested this last method in our lab.

Mounting and unmounting: Correct positioning of the embryos is critical during photoconversion. The mold for making agarose wells facilitates the mounting of embryos such that the anterior-posterior axis of the embryo consistently lies at 25° relative to the surface of the dish. We have found the mold to be applicable for photoconversion at all developmental stages between 36 and 55 hpf. In mounting, the tilt of the embryo in the left-right direction needs to be adjusted according to the embryo's developmental stage and the area one wishes to photoconvert. For example, to photoconvert the AVC at 36 hpf, the embryo should face roughly 15° to the left, while at 55 hpf, the embryo should face roughly 10° to the left. Under the stereomicroscope, you should be able to see the AVC unobscured by the ventricle or the atrium. If, for example, the atrium lies over the AVC, it would not be possible to photoconvert just the AVC or just the atrium. For imaging purposes, the same mold is used to position embryos between 36 and 96 hpf. The mold could be made using a variety of materials using a 3D printer or using traditional mechanical workshop tools. Stainless steel or chemical and heat resistant plastic are both suitable materials. Should the reader wish to create the mold using a 3D printer, please see supplementary information for the .STL file of the mold template.

Adding sufficient LMP agarose to cover the entire embryo is advised to ensure that the embryos do not become dislodged between mounting and photoconversion/imaging, especially if the microscope is not located directly adjacent to the mounting station. In general, provided that the embryos are properly mounted, LMP agarose does not stick to the embryos after unmounting. However, should some LMP agarose remain stuck on the embryo, the LMP agarose can be removed by gently teasing the embryo from the agarose using forceps.

Embryos, especially embryos less than 48 hpf, can be easily damaged during mounting and unmounting. For embryos less than 48 hpf, we recommend fire polishing the end of the glass pipette to prevent damaging embryos during pipetting.

Microscope: This protocol was performed using a Leica SP8 microscope. However, photoconversion is a well-established process and this protocol should be easily convertible to other upright microscopes equipped with lasers capable of producing 405 nm, 488 nm and 561 nm light. For embryos up to 62 hpf, images were acquired using an argon excitation laser and a DPSS 561 laser for green and red Kaede, respectively. For embryos at stages older than 62 hpf, green Kaede can be excited using a multiphoton laser set to 940 nm, which has a greater penetration depth than 488 nm light. In theory, red Kaede should have a two-photon excitation spectra similar to that of DsRed, and could be imaged at 950 nm¹⁷.

An objective lens with a high numerical aperture is necessary to efficiently photoconvert EdCs and to acquire high-resolution images. Since the embryos are kept in media, it is important to choose a microscope equipped with a water objective lens.

Photoconverting the region of interest: It is advisable to be conservative when selecting the ROI to photoconvert - the 405 nm laser diffuses as it travels through tissue and can photoconvert cells in an area greater than that selected. If, for example, you wish to label a cell red but would like the cell directly adjacent to remain green, it may be prudent to select only the part of the cell lying further away from the adjacent cell as your ROI. Since Kaede is expressed in the cytoplasm of EdCs, photoconverted protein will diffuse to parts of the cell not selected by the ROI.

Photobleaching and phototoxicity: The Kaede protein, both its green and red forms, is extremely bright and photobleaching is generally not a limiting factor in cell tracking experiments. However, laser light can impact embryonic development and should be minimized, especially during early stages of development. Signs of phototoxicity include developmental delay and, in more serious cases, improper heart looping and malformed valves.

The power of the violet laser (25 %) and the number of iterations the violet laser scans over the ROI (3-6 times) used in this protocol were determined empirically. At these settings, we saw almost complete conversion of Kaede from its green to red form without noticeable developmental delay. The amount of laser power required in other microscopy setups would depend on the laser's intensity. It should be noted that the red form of Kaede can be bleached by intense 405 nm illumination, so longer photoconversion does not necessarily yield more red fluorescence.

Image analysis: The method proposed for the image analysis is a newly developed process to describe the three-dimensional geometry of the AVC and compare it among embryos. It is intended to be applied to monolayer tubular structures, such as the AVC before 48 hpf and potentially the heart tube, the dorsal aorta and others. It is mainly automatic, even though the segmentation on the cutting planes is performed manually by the user and other steps could potentially require manual correction. The manual segmentation represents the most critical part of the process, since knowledge of the structure geometry and training are needed to achieve good results. After practicing, the time-consumption of this step is considerably reduced.

The protocol described here provides an effective way to track endocardial tissue movements during AVC and atrioventricular heart valve development using the Tg(fli1a:gal4FF^ubs, UAS:kaede) zebrafish transgenic line.

Currently, one of the key limitations of this method is that the violet laser beam is not confined in the axial dimension. This makes targeting of single cells for photoconversion, and hence clonal analyses and single cell tracking, difficult. Two years ago, it was discovered that the photoconvertible protein Dendra2 can be primed converted - the protein can be converted from its green form to its red form by first irradiating the protein with blue light, and then with near-infrared light¹⁸. Microscope setups that exploit this property can photoconvert Dendra2 expressing cells in a spatially confined manner in complex 3D tissue structures while avoiding many of the phototoxicity problems associated with intense near-UV irradiation^18,19,20. With the recent discovery of the molecular mechanism behind this phenomenon, variants of many green-to-red photoconvertible fluorescent proteins, including Kaede, have been engineered so that are also capable of entering a primed intermediate state²¹. To our knowledge, zebrafish transgenic lines where a promoter such as fli1a or kdrl drives the expression of protein Dendra2 or other primed convertible fluorescent proteins in endothelial cells are not yet available. However, lines that express Dendra2 ubiquitously are available and may prove useful in certain applications ^22,23.

Materials

Name	Company	Catalog Number	Comments
Materials
Necessary equipment for raising fish and collecting eggs (see the Zebrafish Book22 for details).
Stereomicroscope
Upright confocal microscope (Equipped with lasers operating at 488 nm, 561 nm, and 405 nm, a tunable multiphoton laser, and a Leica HCX IRAPO L, 25 ×, N.A. 0.95 objective)	Leica	Leica SP8
Heat block	ThermoScientific	88870001
35 mm x 10 mm tissue culture dish	Falcon	353001
6 well plate	Falcon	353046
Petri dish
Forceps
Glass pipette
Mold
Reagents
8 mg/mL Tricaine stock solution
1 M BDM stock solution
UltraPure low melting point agarose	Invitrogen	16520-050
Agarose	Lonza	50004
PTU (1-phenyl-2-thiourea)	Sigma Aldrich	P7629
Embryo medium: 30x stock solution
Software
Matlab equipped with the Image Analysis, Curve Fitting, Bio-Formats Toolboxes	MathWorks
8 mg/mL Tricaine stock solution
200 mg of tricaine powder (Ethyl 3-aminobenzoate methanesulfonic acid)	Sigma-Aldrich	E10521
25 mL Danieau
Adjust to pH 7, aliquot and store at -20 °C
1 M BDM stock solution
1 g BDM	Sigma-Aldrich	112135
10 mL ddH2O
Aliquot and store at -20 °C
Embryo medium: 30x stock solution
50.7 g NaCl	Sigma-Aldrich	7647-14-5
0.78 g KCl	Sigma-Aldrich	7447-40-7
1.47 g Magnesium sulfate	Sigma-Aldrich	7487-88-9
2.1 g Calcium nitrite tetrahydrate	Sigma-Aldrich	13477-34-4
19.52 g HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)	Sigma-Aldrich	7365-45-9
Adjust to pH 7.2 and store at room temperature
Mold
PlasCLEAR resin	Asiga
Plus 39 Freeform Pico 3D printer	Asiga