Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Developmental Biology

Your institution must subscribe to JoVE's Developmental Biology section to access this content.

Fill out the form below to receive a free trial or learn more about access:



Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

Cite this Article

Copy Citation | Download Citations | Reprints and Permissions

Salamon, R. J., Zhang, Z., Mahmoud, A. I. Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging. J. Vis. Exp. (157), e60482, doi:10.3791/60482 (2020).


Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.


The heart has long been considered to be a post-mitotic organ, yet recent evidence demonstrates that cardiomyocyte renewal occurs in the adult human heart at about 1% per year1. However, these low rates of cardiomyocyte turnover are insufficient to replenish the massive loss of tissue that occurs following injury. A heart that has suffered a myocardial infarction will lose around one billion cardiomyocytes, often serving as a prelude to heart failure and sudden cardiac death2,3. With over 26 million people affected by heart failure worldwide, there is an unmet need for therapeutics that can reverse the damages inflicted by heart disease4.

In order to bridge this gap in therapeutics, scientists have begun investigating evolutionarily conserved mechanisms that underlie endogenous regeneration following injury. One model for studying mammalian cardiac regeneration is the neonatal mouse. Within the week following birth, neonatal mice have a robust regenerative response following cardiac damage5. We have previously demonstrated that neonatal mice can regenerate their heart via cardiomyocyte proliferation following an apical resection5. Although this technique can evoke cardiac regeneration in the neonates, the surgery lacks clinical relevance to human heart injuries. In order to mimic a human injury in the neonatal mouse model, we have developed a technique to induce a myocardial infarction through a coronary artery occlusion6. This technique requires surgical ligation of the left anterior descending artery (LAD), which is responsible for delivering 40%–50% of the blood to the left ventricular myocardium6,7. Thus, the surgery results in an infarct that impacts a significant portion of the left ventricular wall. This damage to the myocardium will stimulate cardiomyocyte proliferation and heart regeneration in neonates5.

The coronary artery occlusion surgery provides a highly reproducible and directly translational method to uncover the inner workings of cardiac regeneration. The neonatal surgery parallels coronary artery atherosclerosis in the human heart, where accumulation of plaque within the inner walls of the arteries can cause an occlusion and subsequent myocardial infarction8. Due to a void in therapeutic treatments for heart failure patients, an occlusion in the LAD is associated with mortality rates reaching up to 26% within a year following injury9, and consequently has been termed the "widow maker." Advancements in therapeutics require a model that accurately reflects the complex physiological and pathological effects of cardiac injury. Our surgical protocol for neonatal mouse cardiac injury provides a platform that allows researchers to investigate the molecular and cellular cues that signal mammalian heart regeneration after injury.

Recent research highlights the dynamic relationship between the extracellular environment and proliferating cardiomyocytes. For example, the postnatal regenerative window can be extended by decreasing the stiffness of the extracellular matrix surrounding the heart10. Biomaterials from the neonatal extracellular matrix can also promote heart regeneration in adult mammalian hearts following cardiac injury11. Also accompanying cardiomyocyte proliferation is an angiogenic response12,13; collateral artery formation unique to the regenerating heart of the neonatal mouse was shown to be essential for stimulating cardiac regeneration12. Moreover, our lab has demonstrated that nerve signaling regulates cardiomyocyte proliferation and heart regeneration via modulation of growth factor levels, as well as the inflammatory response following injury14. These findings emphasize the need to trace non-myocyte cell populations in response to cardiac injury. In order to accomplish this goal, we have taken advantage of the Cre-lox recombination system in transgenic mice lines to incorporate constitutive or conditional expression of fluorescent reporter proteins for lineage tracing. Furthermore, we can use advanced methods to determine clonal expansion patterning with the Rainbow mouse line, which relies on stochastic expression of the Cre-dependent, multi-color fluorescent reporters to determine the clonal expansion of targeted cell populations15. Employing lineage tracing with the neonatal coronary artery occlusion surgery is a powerful tool for dissecting the intricate cellular mechanisms of cardiac regeneration.

Tracking the lineage of fluorescently labeled cells with three-dimensional (3D) whole organ imaging is difficult to achieve using traditional sectioning and reconstruction technique – especially when cell populations are fragile, such as nerve fibers or blood vessels. While direct whole-mount imaging of the organ by optical sectioning can capture superficial cell populations, structures that reside deep within the tissue remain inaccessible. To circumvent these barriers, tissue clearing techniques have been developed to reduce the opacity of whole organ tissues. Recently, significant advances have been made to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel (CLARITY)–based methods, which clear fixed tissue via lipid extraction16. Steps are also taken to homogenize the refractive index and subsequently reduce light scattering while imaging17. One such method is active CLARITY, which expedites lipid decomposition by using electrophoresis to penetrate the detergent throughout the tissue18. Although effective, this tissue clearing method requires expensive equipment and can cause tissue damage, making the approach incompatible with fragile cell populations such as the cardiac nerves19. Thus, we employ the passive CLARITY approach, which relies on heat to gently facilitate detergent penetration, therefore aiding in the retention of intricate cell structures20,21.

Passive CLARITY is typically thought to be less efficient than active CLARITY18, as the technique is often accompanied by two major obstacles: the inability to clear the entire organ depth and the extensive amount of time required to clear adult tissues. Our passive CLARITY approach overcomes both of these barriers with an expeditated clearing process that is capable of fully clearing neonatal and adult heart tissue. Our passive CLARITY tissue clearing technique has reached an efficiency that permits the visualization of a variety of cardiac cell populations, including rare populations distributed throughout the adult heart. When the cleared heart is imaged with confocal microscopy, the architecture of cell-specific patterning during development, disease, and regeneration can be illuminated.

Subscription Required. Please recommend JoVE to your librarian.


All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals and in compliance with the Institutional Animal Care and Use Committee in the School of Medicine and Public Health at the University of Wisconsin–Madison. All methods were performed on wild type C57BL/6J (B6) and transgenic mouse lines obtained from Jackson Laboratories.

1. Coronary Artery Occlusion (Myocardial Infarction) Induced via Ligation of the Left Anterior Descending Artery (LAD) in 1-Day-Old Neonatal Mice6

  1. Separate the 1-day-old neonatal pups from the mother by placing them into a clean cage along with some of the original nesting material.
  2. Place half of the cage onto a heating pad set to medium heat. The pups should remain on the unheated side of the cage, only being placed onto the heated side after surgery.
  3. Create a sterile surgical area under an operating microscope. Gather sterilized surgical equipment (Table 1).
  4. Anesthetize the pup via hypothermia: wrap the pup in gauze to avoid direct skin contact with ice and bury it in an ice bed for approximately 3–4 min. Check hypothermia of the mice periodically by performing a toe pinch. Neonates tolerate hypothermia well, however, prolonged exposure to hypothermia may result in frostbite and subsequent mortality.
  5. Once anesthetized, place the mouse onto the surgical area in the supine position, securing the arms and legs with tape. Sterilize the surgical area of the mouse with an antiseptic solution.
  6. Locate the lower chest region and make a transverse incision in the skin with small dissecting scissors. To widen the surgical view of the ribs, separate the skin from the muscle by lifting the skin gently with a pair of dressing forceps and gently press against the intercostal muscles with the small scissors in the closed position.
  7. Locate the fourth intercostal space (Figure 1A) and make a small, superficial puncture using sharp forceps, being careful not to puncture any internal organs. Perform a blunt dissection by widening the area in between the intercostal muscles with dressing forceps. Proper anatomical positioning of the incision is essential for appropriate access to the heart.
  8. Gently guide the heart out of the chest cavity by placing a finger and applying increasing pressure on the left side of the abdomen while holding the intercostal space open with dressing forceps (Figure 1B). Once the heart is outside of the chest, remove the dressing forceps, relieve pressure, and allow the heart to rest on the intercostal muscles.
  9. Locate the LAD as the area of the heart that has less pooled blood and is in the correct anatomical position (Figure 1C). The LAD can be only seen under the microscope if the heart is accessed within a few minutes of beginning surgery.
  10. Perform LAD ligation by suturing the LAD with a 6-0 suture (Figure 1D). Tie a square knot twice to induce myocardial infarction (Figure 1E). The depth of the suture into the myocardium may vary, however, proper anatomical positioning of the LAD ligation is crucial for reproducibility. When ligating the LAD, the suture should be pulled tightly but carefully so as not to sever the LAD. Blanching at the apex of the heart will be seen immediately (Figure 1E)
  11. Allow the heart to slip back into the chest cavity; this process can be gently facilitated with dressing forceps. Suture the ribs together with a surgeon's knot followed by a square knot, using blunt forceps to lift the upper set of ribs while passing a 6-0 suture through the upper and lower set of ribs.
  12. Remove the tape that was used to secure the hind legs of the pup.
  13. Adhere the skin together by placing a small amount of skin glue on the upper abdomen. Then, grab the skin of the lower abdomen with fine forceps and cover the exposed chest region. Limit the amount of excess glue that remains on the pups, as this can increase the likelihood of rejection and cannibalism by the mother.
  14. Immediately facilitate the recovery from anesthesia by placing the pup onto a heating pad set to medium heat. Periodically switch the placement of the neonates to evenly warm all parts of the body.
  15. Allow the neonate to remain directly on the heating pad for 10–15 min. Typically, movement is regained within 5 min of being placed onto heat.
  16. Clean the residual blood and glue with an alcohol wipe.
  17. Cover foreign scents on neonates by rubbing the entire body with bedding from the original cage. Place the pup into the cage on the heated side while other surgeries are being performed.
  18. Once all surgeries are completed and pups are warm and mobile, transfer the litter along with the original nesting material to the mother's cage.
  19. Monitor the mice for 30–60 min after surgery and watch for the mother's acceptance of the pups by nesting and/or grooming.
  20. Check on the mice the morning following surgery. If mother is distressed and has not nested the pups, consider a foster mother for the pups.

2. Clearing the Mouse Heart with Passive CLARITY21,22,23

  1. Anesthetize the mouse with isoflurane. Perform a toe pinch to ensure the mouse is fully sedated.
  2. Place the mouse onto a clean, surgical area in the supine position, securing the arms and legs with tape.
  3. Maintain isoflurane sedation on the mouse using a nose cone until the heart is extracted.
  4. Open the lower chest by holding the fur just below the xiphoid process with tissue forceps and make an incision spanning the width of the ribcage using the large dissecting scissors.
  5. Cut alongside of the distal portions of the rib cage with surgical scissors.
  6. Expose the diaphragm muscle by grasping the xiphoid process with tissue forceps. Detach the diaphragm using curved forceps.
  7. While maintaining a grasp of the xiphoid process, pull the tissue cranially until the beating heart is accessible.
  8. Grasp the heart at the base with curved forceps and dissect the heart from the chest cavity by cutting the aorta and superior vena cava with iridectomy scissors.
  9. While the heart is still beating, place the heart into a Petri dish filled with PBS so that the heart pumps out the blood inside as it keeps beating. Myocardial infarction can be confirmed by checking that the placement of the suture is in the proper anatomical position for LAD ligation.
  10. Gently squeeze the heart with forceps to allow the heart to expel the residual blood.
  11. Transfer the mouse heart into a disposable 2.5 mL glass shell vial with 2 mL of PBS. Wash away residual blood by incubating the heart on a shaker for 10 min at room temperature (RT) several times. Change the PBS solution each time until PBS remains clear.
  12. Discard the PBS and fill the vial with 2 mL of cold 4% paraformaldehyde (PFA). Incubate for 4 hours at RT (Figure 2A).
  13. After incubation, discard the PFA and the vial with 2 mL of PBS. Wash the heart on a shaker for 10 min at RT. Repeat the washing step twice, draining and filling the vial with new PBS each time to fully wash away excess PFA.
  14. Discard PBS and fill the vial with 2 mL of 4% acrylamide and 0.5% VA-044 solution. Incubate overnight at 4 °C.
  15. The next day, perform polymerization by transferring the vial to a heat block set at 37 °C for 3 hours.
  16. Transfer the heart into a new glass shell vial and repeat step 2.12 (PBS wash cycle).
  17. Discard PBS and fill the vial with 2 mL of Clearing Solution (Table 2). Incubate at 37 °C until the heart is cleared. Change out the solution and refill with fresh Clearing Solution every 2–3 days. The clearing process could take up to several weeks (Figure 2B-C).
    NOTE: P1 hearts typically take around 3–5 days, whereas P21 hearts can take nearly a month before Clearing Solution incubation is complete.
  18. Discard PBS and fill the vial with 2 mL of PBS and repeat step 2.12 (PBS wash cycle). Refill the vial with fresh PBS and incubate overnight at 37 °C.
  19. If immunostaining will be performed, skip steps 2.21–2.22 and proceed to Section 3 for immunostaining. If relying solely on endogenous fluorescence, proceed with steps 2.21–2.22 and skip Section 3.
  20. Discard PBS and change the solution to Refractive Index Matching Solution (RIMS) (Table 3). Incubate overnight at 37 °C.
  21. After incubation, the cleared tissue can be stored in RIMS solution at RT. Tissue may appear to be white and opaque in the center when first transferred into RIMS; tissue should become transparent after being incubated in RIMS at room temperature for several weeks (Figure 2D).

3. Optional: Immunohistochemistry Staining of the Whole-Mount Mouse Heart

  1. Remove the cleared heart from the RIMS solution and place into a clean 2.5 mL glass vial with 2 mL of PBST (PBS with 0.1% Triton-X 100)
  2. Wash the heart in PBST 3 times on an orbital rotator with 30 min incubations at RT.
  3. Block non-specific antibody binding by immersing the heart in 2 mL of 20% blocking buffer (diluted in PBST) and incubate with rotation for 3 hours at RT. Transfer to 4 °C to stain with rotation overnight.
    NOTE: Blocking buffer is made from normal serum matching the species in which the secondary antibody was raised.
  4. Wash the heart in PBST 3 times with rotation for 5 min incubations at RT.
  5. Immerse the heart in primary antibody (diluted in 2% blocking buffer with PBST) and prevent light exposure by wrapping the glass vial in aluminum foil. Incubate with rotation overnight at RT.
    NOTE: From this point forward, aluminum foil should be continuously used to protect the secondary from ambient light exposure.
  6. Incubate for an additional 24 hours with rotation at 4 °C.
  7. Following primary staining, repeat step 3.2 (long PBST wash cycle) to remove the unbound primary antibody from the tissue. Extend the wash with an overnight incubation with rotation at RT.
  8. Working in an area with limited lighting to prevent secondary antibody light exposure, immerse the heart in secondary antibody (diluted in 2% blocking buffer with PBST) and incubate with rotation for 3 hours at RT. Transfer to 4 °C to stain with rotation overnight.
  9. On the next day, repeat step 3.2 (long PBST wash cycle) to remove unbound secondary antibody.
  10. Replace the solution with 2% blocking buffer (diluted in PBST). Remove residual unbound antibody by washing overnight with rotation at RT.
  11. The next day, check that excess secondary antibody has been removed using confocal microscopy. Extend the wash as needed, replacing the 2% blocking buffer solution daily. Proceed once little to no non-specific secondary is present.
  12. Store the immunostained heart in PBS at 4 °C.
  13. Directly before whole-mount microscopy, incubate the heart in RIMS solution overnight at 37 °C. Extend the incubation an additional 24 hours if the tissue is still not fully cleared.
  14. Store the fully cleared and immunostained heart in RIMS at RT.

4. Visualizing Non-myocyte Populations in 3D with Single-Photon Confocal Microscopy Imaging of the Cleared Mouse Heart

NOTE: If mouse hearts are harvested embryonically, continue with section 4.1. For mouse hearts harvested postnatally, continue with section 4.2.

  1. Imaging the Cleared Embryonic Mouse Heart
    1. Fill the microscope depression slide with the PBS solution.
    2. Carefully pick up the cleared heart with curved forceps and place the tissue onto the slide.
    3. Mount the slide with a glass coverslip. The tissue can now be imaged under a confocal microscope.
  2. Imaging the Cleared Postnatal Mouse Heart
    1. Fill half of the chamber of the depression slide with PBS solution. In order to create a chamber large enough to fit an adult mouse heart, a 3D-printed polypropylene depression slide was custom-made (Figure 4).
    2. Carefully pick up the cleared heart with curved forceps and place the tissue into the chamber. Fill the remaining volume of the chamber with PBS.
    3. Fill the chamber with PBS so the surface of the liquid forms a dome above the top of the chamber. Mount the cover slide. The tissue can now be imaged under an upright confocal microscope.

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

Often the two most challenging steps are guiding the heart out of the chest cavity and ligating the LAD. To troubleshoot these steps, adjustments may be made in the placement of the initial puncture between the fourth intercostal muscles; if the puncture and blunt dissection are too close in proximity to the sternum, the heart may not be able to exit the chest cavity (Figure 1A).

Additionally, increased pressure on the left abdomen may be needed to facilitate this process (Figure 1B). Complications may also occur when resting the heart on the intercostal muscles. We found the heart will remain outside of the cavity without withdrawing when the blunt dissection is kept to a minimal size and performed mainly in the horizontal direction. This orientation also allows for clear visualization and accessibility to the LAD (Figure 1C).

When placing the suture needle behind the LAD, it is suggested to penetrate deep into the anterior wall of the left ventricle, as a superficial ligation has less room for adjustment in the final suture placement (Figure 1D). Tying the suture around the LAD should be performed with controlled, steady movements, as the LAD is fragile and severing this coronary artery and the anterior wall will cause mortality (Figure 1E). Within 5–10 minutes after surgery is complete, the neonate should be lively and breathing normally.

When proceeding to the passive CLARITY protocol, hearts harvested from a mouse line that incorporates an endogenous fluorescent reporter for cell lineage tracing should be protected from light (Figure 2), which can be accomplished by wrapping the glass vial in aluminum foil. During the clearing step, the time incubating in Clearing Solution is variable depending on the age of the mouse when the heart was harvested. This step is complete when the entire tissue is consistently opaque, with no discoloration in the center (Figure 2B-C). The hearts will become increasingly transparent after storage in RIMS solution at room temperature for a couple of days (Figure 2D). It should be noted that tissue expansion may occur during the clearing process.

Our passive CLARITY protocol permits effective antibody penetration and thus is compatible with immunohistochemistry methods to label protein(s) of interest. This was validated in Actl6bCre;Rosa26tdT transgenic mice, which label mature neurons with the tdTomato (tdT) reporter protein. The cardiac nerves are mainly superficial, with some populations residing in the epicardial layer, therefore we used this cell population as a proof-of-principal model for the reproducibility of endogenous reporter signal (Figure 3A), as well as preservation of reporter protein conformation before and after CLARITY (Figure 3B-C). Our representative results from Actl6bCre;Rosa26tdT mice show that the endogenous tdT protein signal seen in nerves of an uncleared P7 heart was faithfully recapitulated in nerves of both uncleared and cleared tdT immunolabeled P7 hearts (Figure 3). To immunolabel tdT-positive nerves, a Red Fluorescent Protein (RFP) primary antibody (rabbit polyclonal; 1:200 dilution; Rockland #600-401-379) was used along with an Alexa Fluor 488 secondary antibody (goat polyclonal; 1:1000 dilution; Invitrogen #A-11008). To visualize the cleared heart by eye during immunostaining and imaging steps, an ultraviolet flashlight may be briefly used to illuminate the heart.

Figure 1
Figure 1: Induction of Myocardial Infarction in the Neonatal Mouse through Coronary Artery Occlusion of the Left Anterior Descending Artery (LAD). (A) The fourth intercostal space, indicated by a single asterisk (*), is located and a blunt dissection is performed. (B) Pressure is applied on the left abdomen to guide the heart out of the chest cavity. (C) The LAD, marked by a double asterisk (**), is identified as being the predominant artery and by anatomical position. (D, E) A suture is then passed behind the LAD and a square knot is tied around the LAD to induce a coronary artery occlusion and subsequent infarct. (E) Once complete, blanching can be seen below the suture, at the apex of the heart. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Progression of the Passive CLARITY Method on a P14 Mouse Heart. Hearts were harvested from P14 mice, (A) fixed, and (B–D) underwent the passive CLARITY protocol. For hearts taken at a P14 timepoint, Clearing Solution incubation step is (B) incomplete when discoloration at the center of the heart is apparent, seen after 6 days, and (C) complete when heart appears evenly opaque, seen after 10 days. (D) After the heart is stored in RIMS, the tissue is completely cleared. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Whole-Mount 3D Imaging of P7 Mouse Hearts with Confocal Microscopy. Representative whole-mount 3D images from P7 Actl6bCre;Rosa26tdT transgenic mice hearts are shown as maximum intensity projections of z-stacked images. Hearts were imaged to show (A) endogenous tdTomato (tdT) fluorescence directly after harvesting (red) (B) immunostaining of tdT-positive nerves in an uncleared heart (Alexa Fluor 488; green) and (C) reproducible immunolabeling of tdT-positive nerves in a cleared heart (Alexa Fluor 488; green). Please click here to view a larger version of this figure.

Figure 4
Figure 4: 3D Printed Polypropylene Depression Slides. To image the postnatal heart samples, custom depression slides were 3D printed on polypropylene. The slide dimensions are 25 mm x 75 mm x 1 mm, with a slide depression well 13 mm in diameter and either (A) 6.5 mm or (B) 17 mm in depth. Please click here to view a larger version of this figure.

Equipment Type Description Company Catalog Number
Glass Vial 12 x 35mm Vial with Cap Fisherbrand 03-339-26A
6-0 Prolene Sutures Polypropylene Sutures Ethicon 8889H
Sharp Forceps Fine Tip, Straight, 4.25 in Sigma-Adrich Z168777
Dressing Forceps Dissecting, 4.5 in Fisherbrand 13-812-39
Needle Holder Mayo-Hegar, 6 in Fisherbrand 08-966
Small Dissecting Scissor 30 mm Cutting Edge Walter Stern Inc 25870-002
Tissue Forceps Medium Tissue, 1X2 Teeth Excelta 16050133
Large Dissecting Scissors Straight, 6 in Fisherbrand 08-951-20
Iridectomy Scissors 2 mm Cutting Edge Fine Science Tools 15000-03
Curved Forceps Half Curved, Serrated, 4 in Excelta 16-050-146

Table 1: Surgical Equipment.

Clearing Solution
Reagent Final Amount Notes
Sodium Dodecyl Sulfate (SDS) 8.0% w/v 40 g
Boric acid 1.25% w/v 6.25 g
1-thioglycerol 0.5% w/v 5 μL/mL Added as needed to solution
Add 400 ml of ultrapure H20 to 1 L beaker. Mix in SDS and Boric Acid with magnetic stirring.
Adjust pH to 8.5 using 6M NaOH. Bring volume to 500 ml and Autoclave.
Store solution at room temperature.
Other CLARITY Reagents
Reagent Final Stock Notes
PFA 4.0% 16% Diluted in PBS
Acrylamide 4.0% 40% Diluted in PBS
VA-044 0.5% 10% Added as needed to solution

Table 2: Clearing Solution and Other CLARITY Reagents.

Reagent Final Amount
Phosphate Buffer 0.02 M 90 mL
Histodenz 133.3% w/v 120 g
Sodium Azide 0.05% w/v 45 mg
Add 90 mL of 0.02 M Phosphate Buffer to 250 mL glass media bottle wrapped in aluminum foil.
Mix in reagents listed with magnetic stirring. Allow components to dissolve overnight.
Vortex solution and filter purify with filtration system into a sterile 250 mL glass media bottle.
Wrap bottle in aluminum foil and store solution at room temperature.

Table 3: Refractive Index Matching Solution (RIMS).

Subscription Required. Please recommend JoVE to your librarian.


Cell-cell interactions between cardiomyocytes and non-myocyte populations are a determining factor of whether the heart will undergo fibrosis or repair following injury. Discoveries have been made demonstrating that a variety of cell types, including nerves14, epicardial cells24, peritoneal macrophages25, arterioles12,13, and lymphatic endothelial cells26, all play an essential role in mediating cardiac repair. These cell lineages and others of interest can be genetically traced during development, disease, and regeneration by applying Cre-lox and CRISPR-Cas9 technologies in mice. When coupled with organ clearing and advanced microscopy methods, the contributions of non-myocyte populations can be accurately assessed, opening the door to elucidate cellular and molecular targets of myocardial regeneration following injury.

The efficiency of the protocol is dependent on consistent and reproducible ligation of the LAD during coronary artery occlusion surgery. Neonatal mice are sensitive to extended exposure to hypothermia; thus, the surgery must not only be performed with accuracy but also within minutes. From start to finish, the myocardial infarction surgery should take less than 8 minutes. We recommend first practicing on several litters of the same background as the experimental mice until proficiency is achieved.

Progression of cardiac repair can be assessed by using echocardiography to measure cardiac function (i.e. fractional shortening, ejection fraction, systolic and diastolic volume) once within 3 to 7 days after surgery and again shortly before harvesting the heart, suggested between 21- and 28-days post injury. Hearts can be collected for clearing at multiple timepoints following myocardial infarction surgery.

The clearing step (step 2.17) is subject to variation in duration depending on age and strain of the mouse from which the heart was harvested, which can result in differences in heart size. For a B6 background mouse, clearing duration based on age is estimated as follows: P1 (7-10 days), P7 (14-17 days), P14 (21-24 days), P21 (28-31 days), P28 (35-38 days). Although the primary focus of our laboratory is heart clearing, our passive CLARITY method has been successful for clearing mice lungs (unpublished results) and we foresee no limit on broadly applying this to other organs. Overall, our expedited clearing process is highly valued for the ability to clear tissues rapidly and effectively.

It should be noted that complications can arise when applying tissue clearing techniques in organs with endogenous reporters in rare subpopulations. Reporter signal in dense cell populations (such as myocytes23) seem to be more resistant to the clearing process and thus the signal is typically retained; however, other more sensitive cell populations (such as cardiac nerves) are prone to having endogenous fluorescent protein signal quenched after prolonged fixation or clearing. This became apparent in the Actl6bCre;Rosa26tdT reporter mouse model, where P7 hearts fixed briefly for 15 minutes showed strong tdT signal (Figure 3A), however this fluorescent signal was quenched after fixation for 1 hour or overnight incubation in the Clearing Solution (data not shown). In the scenario of lost reporter signal, antibody staining targeting the reporter protein is compatible with tissue clearing to amplify the signal (Figure 3). The addition of an immunolabeling step can be advantageous for tissues undergoing extensive imaging, as conjugated antibodies are bleach-resistant and produce stable, long-term expression. With the ability to track proteins endogenously and through immunostaining, our protocol uniquely allows for precise localization of rare cardiac cell populations that reside deep within the heart.

Cleared hearts can undergo lineage tracing or fluorescent protein expression analysis using whole-mount 3D confocal microscopy. The confocal microscope is equipped with laser lines optimized to excite florescent reporters (or conjugated secondary antibodies), for example: BFP (DAPI, Alexa Fluor 405), EGFP (FITC, Alexa Fluor 488), DsRed (TRITC, Alexa Fluor 546/555), and APC (Cy5, Alexa Fluor 647) at 405 nm, 488 nm, 561 nm, and 683 nm, respectively. For heart samples unable to fit in a depression slide – common if the heart harvested postnatally – a custom depression slide can be made by 3D printing on polypropylene. Custom slides followed dimensions of a microscope depression slide (25 mm x 75 mm x 1 mm), varying the well depth to either 6.5 mm or 17 mm (Figure 4).

In order to visualize the reporter cell lines in 3D, the confocal microscope is set to acquire z-stacked images throughout the heart. When imaging larger hearts, the entire heart may not be able to be captured in a single z-stacked image even with a low magnification objective. In this scenario, a series of multiple z-stacked images taken at different heart regions can be stitched together using a large image function. This is accomplished by calibrating the microscope to the appropriate objective lens and specifying the field for the large image scan area. Then, z-stacked images undergo 3D reconstruction using a volume rendering program and maximum intensity projection (Figure 3). The high-resolution images acquired can be analyzed to determine precise 3D spatial cell patterning of targeted cell populations.

Collectively, this protocol provides a powerful molecular tool to understand the dynamic cellular changes that occur during cardiac repair and regeneration. This method describes the steps to induce a myocardial infarction, perform whole organ clearing, trace cell specific populations, and analyze 3D cell patterning. Together, these techniques provide access to trace cell populations that were previously inaccessible due to their sparse presence or location within the tissue. This will enable further investigation of questions paramount to advance therapeutic approaches in regenerative medicine, particularly those aimed at stimulating endogenous heart regeneration.

Subscription Required. Please recommend JoVE to your librarian.


The authors declare that they have no competing financial interests.


Funding for this project was provided by the UW School of Medicine and Public Health from the Wisconsin Partnership Program (A.I.M.), and an American Heart Association Career Development Award 19CDA34660169 (A.I.M.).


Name Company Catalog Number Comments
6-0 Prolene Sutures Ethicon 8889H Polypropylene Sutures
Boric acid
Curved Forceps Excelta 16-050-146 Half Curved, Serrated, 4 in
Dressing Forceps Fisherbrand 13-812-39 Dissecting, 4.5 in
Glass Vial Fisherbrand 03-339-26A 12 x 35 mm Vial with Cap
Histodenz Sigma-Aldrich Density gradient medium
Iridectomy Scissors Fine Science Tools 15000-03 2 mm Cutting Edge
Large Dissecting Scissors Fisherbrand 08-951-20 Straight, 6 in
Needle Holder Fisherbrand 08-966 Mayo-Hegar, 6 in
Phosphate Buffer
Sharp Forceps Sigma-Adrich Z168777 Fine Tip, Straight, 4.25 in
Small Dissecting Scissor Walter Stern Inc 25870-002 30 mm Cutting Edge
Sodium Azide
Sodium Dodecyl Sulfate (SDS)
Tissue Forceps Excelta 16050133 Medium Tissue, 1X2 Teeth
VA-044 Wako Chemicals Water-soluble azo initiator



  1. Lazar, E., Sadek, H. A., Bergmann, O. Cardiomyocyte renewal in the human heart: insights from the fall-out. European Heart Journal. 38, (30), 2333-2342 (2017).
  2. Kikuchi, K., Poss, K. D. Cardiac regenerative capacity and mechanisms. Annual Review of Cell and Developmental Biology. 28, 719-741 (2012).
  3. Habecker, B. A., et al. Molecular and cellular neurocardiology: development, and cellular and molecular adaptations to heart disease. The Journal of Physiology. 594, (14), 3853-3875 (2016).
  4. Savarese, G., Lund, L. H. Global Public Health Burden of Heart Failure. Cardiac Failure Review. 3, (1), 7-11 (2017).
  5. Porrello, E. R., et al. Transient regenerative potential of the neonatal mouse heart. Science. 331, (6020), 1078-1080 (2011).
  6. Mahmoud, A. I., Porrello, E. R., Kimura, W., Olson, E. N., Sadek, H. A. Surgical models for cardiac regeneration in neonatal mice. Nature Protocols. 9, (2), 305-311 (2014).
  7. Karwowski, J., et al. Relationship between infarct artery location, acute total coronary occlusion, and mortality in STEMI and NSTEMI patients. Polish Archives of Internal Medicine. 127, (6), 401-411 (2017).
  8. Lusis, A. J. Atherosclerosis. Nature. 407, (6801), 233-241 (2000).
  9. MAGGIC. The survival of patients with heart failure with preserved or reduced left ventricular ejection fraction: an individual patient data meta-analysis. European Heart Journal. 33, (14), 1750-1757 (2012).
  10. Notari, M., et al. The local microenvironment limits the regenerative potential of the mouse neonatal heart. Science Advances. 4, (5), 5553 (2018).
  11. Porrello, E. R., et al. Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family. Proceedings of the National Academy of Sciences of the United States of America. 110, (1), 187-192 (2013).
  12. Das, S., et al. A Unique Collateral Artery Development Program Promotes Neonatal Heart Regeneration. Cell. 176, (5), 1128-1142 (2019).
  13. Wang, Z., et al. Decellularized neonatal cardiac extracellular matrix prevents widespread ventricular remodeling in adult mammals after myocardial infarction. Acta Biomateria. 87, 140-151 (2019).
  14. Mahmoud, A. I., et al. Nerves Regulate Cardiomyocyte Proliferation and Heart Regeneration. Developmental Cell. 34, (4), 387-399 (2015).
  15. Yanai, H., Tanaka, T., Ueno, H. Multicolor lineage tracing methods and intestinal tumors. Journal of Gastroenterology. 48, (4), 423-433 (2013).
  16. Ariel, P. A beginner's guide to tissue clearing. The International Journal of Biochemistry & Cell Biology. 84, 35-39 (2017).
  17. Chung, K., et al. Structural and molecular interrogation of intact biological systems. Nature. 497, (7449), 332-337 (2013).
  18. Epp, J. R., et al. Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs. eNeuro. 2, (3), (2015).
  19. Lee, H., Park, J. H., Seo, I., Park, S. H., Kim, S. Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine. BMC Developmental Biol. 14, 48 (2014).
  20. Wan, P., et al. Evaluation of seven optical clearing methods in mouse brain. Neurophotonics. 5, (3), 035007 (2018).
  21. Phillips, J., et al. Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease. Scientific Reports. 6, 26013 (2016).
  22. Blom, J. N., Lu, X., Arnold, P., Feng, Q. Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration. Journal of Visualized Experiments. (111), e54100 (2016).
  23. Sereti, K. I., et al. Analysis of cardiomyocyte clonal expansion during mouse heart development and injury. Nature Communications. 9, (1), 754 (2018).
  24. Lepilina, A., et al. A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell. 127, (3), 607-619 (2006).
  25. Wang, J., Kubes, P. A Reservoir of Mature Cavity Macrophages that Can Rapidly Invade Visceral Organs to Affect Tissue Repair. Cell. 165, (3), 668-678 (2016).
  26. Vieira, J. M., et al. The cardiac lymphatic system stimulates resolution of inflammation following myocardial infarction. Journal of Clinical Investigation. 128, (8), 3402-3412 (2018).



    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Usage Statistics