A Microscopic 2,3,5-Triphenyltetrazolium Chloride Assay for Accurate and Reliable Analysis of Myocardial Injury

Summary

The present 2,3,5-Triphenyltetrazolium Chloride (TTC) assay introduces a refined TTC staining protocol that enables the analysis of myocardial injury at microscopic resolution, producing high-quality images that allow for accurate and reliable assessment of infarct size and cardiomyocyte viability at the cellular level.

Abstract

In this protocol, we present a refined 2,3,5-Triphenyltetrazolium Chloride (TTC)-based method for the accurate and reliable analysis of myocardial infarction (MI) at cellular resolution. This protocol involves a two-step TTC staining procedure performed on infarcted hearts 24 hpost-MI: perfusion, in which a 1% TTC solution is retrogradely perfused through the coronary vasculature via an aortic cannula, and immersion, where the heart is subsequently incubated in TTC solution at 4 °C overnight. After staining, the heart is embedded in OCT compound and cryosectioned transversely into 50-µm thick slices at 100-µm intervals. Following a 24 h fixation process with Zamboni's fixative, a sharp and distinct boundary between viable myocardium (deep red) and necrotic tissue (yellow) becomes discernible under microscopic examination. Notably, this microscopic TTC assay enables MI-detection not only in the acute phase (24 h post-MI) but also across an extended time window beyond 7 days. Because the microscopic TTC assay generates multiple-layer images (80 slices per heart), we developed a robust, color-based semi-automated algorithm that enables rapid and efficient analysis of large image datasets and quantifies the global infarct size within minutes. With these enhancements, the current approach substantially refines the TTC staining process and provides a broadly applicable and valuable tool for laboratories investigating cardiac ischemic injury.

Introduction

Over the past two decades, cardioprotection has continued to be pursued as a key strategy for preserving cardiomyocytes following severe ischemic insults to the heart¹. In both experimental and clinical settings, the final extent of myocardial injury is a critical metric for evaluating the efficiency of cardioprotective interventions². Thus, a reliable and accurate assessment of infarct size is highly desirable and fundamentally important.

In rodent models, the standard protocol for assessing postmortem infarct size primarily relied on the activity of mitochondrial dehydrogenase, which enzymatically converts the colorless compound 2,3,5-triphenyltetrazolium chloride (TTC) into a red precipitate in viable cells, while it remains light grey in necrotic tissue³. Although TTC-based staining is simple and cost-effective, it presents several methodological challenges and intrinsic limitations. These include cardiac slices undergoing contracture in TTC solution, which badly distorts the tissue and prevents the slices from lying flat for planimetry; uneven slice thickness during sample processing; and poor image quality that hampers the accurate delineation of the infarct boundary. In recent years, several technical modifications have been introduced to the sample preparation process, such as the use of acrylic heart matrices⁴ or semi-freezing the heart to facilitate slicing³. Although these modifications substantially enhanced the practicality of tissue slicing, the conventional approach continues to rely on gross imaging of thick heart sections, often producing variable and suboptimal image quality⁵, with insufficient color contrast that can introduce substantial subjectivity to infarct measurement⁶. Moreover, the conventional method requires the use of whole-heart tissue, which prevents multipurpose analyses on the same tissue samples, such as immune co-staining of regulatory proteins to identify key signaling cascades involved in cardiac survival. As a result, substantial numbers of animals are often required to adequately address various research questions.

To overcome these limitations⁵, we developed a protocol suitable for microscopic imaging of the TTC staining. This method combines "perfusion + immersion" staining steps with cryosectioning of the heart tissue into thin slices optimized for imaging by a light microscope. Although this method was recently reported⁷, we believe that providing detailed experimental procedures enhances its practicality and accessibility. Moreover, because the microscopic TTC method generates high-resolution images comprising multiple heart-section layers, we developed a robust, color-based semi-automatic algorithm to address the time-intensive nature of analyzing large image datasets. The present method enables rapid detection of infarct regions and quantification of global infarct size within minutes.

Overall, the protocol presented here serves as a valuable tool, enabling researchers to efficiently implement this method with ease and accuracy.

Protocol

All experiments were approved by the LANUV of Nordrhein-Westfalen, Germany, and conducted in accordance with the ARRIVE guidelines and the NIH Guide for the Care and Use of Laboratory Animals. All necessary equipment and chemicals are listed in the Table of Materials.

1. Setting up a Langendorff retro-perfusion system

NOTE: The system consists of a two-gang, three-way stopcock connected to an aortic cannula made from a 20-gauge blunted needle and two infusion ports, one for the washing solution (phosphate-buffered saline (PBS) port) and the other for the TTC infusion (TTC port). By alternating the direction of the three-way stopcock, the perfusate can be delivered to the heart interchangeably.

1. Secure the apparatus horizontally on a 150-mm Petri dish using a piece of adhesive tape to prevent unwanted movement during later steps.
2. Adjust the tip of the cannula to be 1 mm above the bottom, aligning it with the level of the aortic root when the heart is placed flat.
3. Prefill the system with ice-cold PBS using a 10-mL syringe through the PBS port.
4. Prepare fresh staining solution (1% TTC dissolved in PBS); a minimal volume of 6 mL is required for each heart.
   CAUTION: TTC is generally considered hazardous and requires careful handling as it may cause mild skin irritation, serious eye irritation, and is harmful if inhaled. Dispose of waste and residues in accordance with local authority requirements.
5. Use a 3-mL syringe to prefill the TTC port with the staining solution.
6. Ensure there are no unintentional air bubbles in the system, as they could cause potential air emboli that would interfere with subsequent staining. In most cases, air bubbles are visible in the system; when present, remove them by withdrawing and refilling PBS while gently tapping the three-way stopcock in the upright cannula position.
7. Form a loose half-square knot using a 6-0 silk suture and place it on the distal end of the cannula for later fixation of the aortic root.

2. Heart sample preparation

NOTE: C57BL/6J mice (20-25 g) were used in this study. Experimental myocardial infarction (MI) was induced by surgically ligating the left anterior descending artery (LAD) for 30-60 min, as described below. The MI induction methodology has been previously reported⁸ and is not detailed in this manuscript. Unless otherwise stated, heart samples were collected 24 h post-MI.

1. Take the MI mouse out of the cage and euthanize it by cervical dislocation under deep isoflurane anesthesia (3 %, v/v).
   NOTE: All euthanasia procedures must be properly documented and reported, including the reason for euthanasia and the method used, in accordance with local regulations .
2. Transfer the euthanized animal to the operating table and secure all four extremities with adhesive tape.
3. Remove pelt from the mid-abdomen to mid-forepaw region using micro-dissecting forceps and scissors.
4. Make a 2-cm transverse incision into the abdominal muscle wall, just below the diaphragm, to allow subsequent thoracotomy.
5. Spread the diaphragm with fine scissors to expose the heart, then make bilateral incisions through the skin, muscle, and ribs to open the thoracic cavity.
6. Cut the diaphragm back bilaterally and fully expose the heart by lifting the sternum.
7. Immediately pour 20 mL of ice-cold PBS onto the heart surface to arrest the heartbeat.
8. Excise the heart along with the surrounding tissues (lungs and esophagus) and transfer the tissue mass into a 50-mL beaker containing ice-cold PBS for about 1 min to allow the heartbeat to subside.
9. Transfer the heart onto a 150-mm Petri dish and trim the aortic root free of connective tissue under a binocular microscope using fine forceps and scissors.
10. Gently remove the thymus and expose the ascending aorta.
11. Trim the aortic root by dissecting the connective tissue.
12. Pick up the aortic stump with fine tweezers and mount it over the cannula that is already connected to a perfusion apparatus prefilled with PBS and TTC staining solution. Use fine tweezers to stabilize the aortic root wall during insertion.
13. Secure the aorta using the pre-formed loose knot on the cannula. Carefully inspect the cannula and the heart while it is being retro-perfused with PBS. Filling of the heart can be visually confirmed by the bulging of the ventricle as the volume of PBS entering the heart increases.
14. Leave the extra-cardiac tissues (lungs and esophagus) in situ to support the heart in a natural and appropriate position.

3. TTC staining

1. Cover the heart with moist tissue paper once the cannulation is secured.
2. Begin rinsing the heart with a pre-connected 10-mL syringe filled with ice-cold PBS at a rate of 2-3 mL/min for 3 min, until the cardiac eluate drains clear. This ensures that only a minimal amount of blood remains in the heart and coronaries. During this step, avoid excessive pressure during perfusion as it can lead to rupture of the infarcted ventricle.
3. Inspect the heart under a binocular microscope to ensure there is no leakage at the aortic root and that it inflates properly under perfusion pressure.
4. Turn the three-way stopcock toward the TTC solution and slowly infuse 1 mL of staining solution through the TTC port over 30 s.
5. Re-infuse 1 mL of TTC solution 5 min later and repeat this step once more after another 5 min (10 min after the first TTC infusion). A total of 3 mL of TTC solution is required for 3x infusion over 15 min.
6. Inspect the heart surface under a binocular microscope. At this point, a clear distinction between the viable myocardium (deep red) and necrotic tissue (light gray) should already be visible.
7. Remove all non-cardiac tissues using fine scissors and transfer the stained heart into a 15-mL conical tube containing 3 mL of TTC solution.
8. Place the conical tube at 4 °C overnight for a second round of staining by immersion.

4. Tissue block preparation

1. Take the heart out of the conical tube and carefully dry it with tissue paper. At this point, staining in the viable myocardium becomes more pronounced, and non-perfused myocardium (epicardial and potentially endocardial regions) also shows light red staining.
2. Fill a blunted, curved 20-gauge cannula with optimal cutting temperature (OCT) compound using a 1-mL syringe.
3. Excise the tricuspid valve with fine scissors and insert the cannula into the right ventricle.
4. Fill the cavity with OCT compound (approximately 50-80 µL) and stop the infusion when backflow is observed.
5. Expose the mitral valve and insert the cannula through the left atrium and the mitral valve into the left ventricle.
6. Gently infuse the cavity with OCT compound (approximately 80 µL) and stop the infusion when outflow from the aorta is observed.
7. Prepare a small cylindrical capsule (1 cm in diameter) made from aluminum foil and fill it with 300 µL of OCT compound.
8. Carefully place the heart into the capsule in an apex-down position and adjust it to a vertical orientation. From the top-down view, the apex should not excessively protrude toward either side.
9. Immerse the capsule in a 2-methylbutane solution pre-chilled to -30 °C to -40 °C.
10. Allow the sample to remain in the solution for 5-10 min to solidify before forming a tissue block.
11. Label the tissue block and store it at -80 °C for further histological analysis.

5. Cryosectioning and fixation

1. Mount the tissue block onto a cryostat chuck using OCT compound.
2. Place the chuck into the cryostat and set the temperature at approximately -24 °C.
3. Slice the tissue block until the apex is visible.
4. Set the section thickness to 50 µm and collect every second slice, resulting in 100-µm intervals between slices.
   NOTE: Avoid tissue folding during cryosectioning. This is largely minimized by filling the empty ventricular cavities with OCT compound (see steps 4.4-4.6).
5. Align the slices sequentially on glass microscope slides, placing eight pieces per slide. In total, 80 sections, i.e., 10 slides, are expected per heart.
6. Dry the slides with a hairdryer on the lowest setting using room-temperature airflow (no heating is needed) for approximately 10 min.
7. Submerge the slides in a staining chamber filled with Zamboni's solution (a fixative containing 2% paraformaldehyde and 0.4 % picric acid, w/v).
   CAUTION: Zamboni's fixative may cause an allergic skin reaction and is suspected of causing genetic defects and carcinogenesis. Dispose of waste and residues in accordance with local authority regulations.
8. Fix the heart tissue at 4 °C overnight. This long-term fixation ensures the heart tissue surface remains free of air bubbles formed during the fixation process.
9. After fixation, take the slides out of the fixative and rinse them three times with PBS.
10. Mount the slides with mounting medium and cover the sections with coverslips. After this step, the heart slides are ready for image acquisition.
11. Store the samples in a slide box at room temperature.
    ​NOTE: As formazan precipitates are water-insoluble, staining can be preserved for years without any loss of intensity.

6. Imaging and processing

1. Set up a light microscope using a 1.25x objective and run CellSens software for imaging.
2. Position the heart section in the center of the field of view.
3. Adjust the camera's exposure time, focus, and white balance as needed.
4. Capture images of each section and save all images from the same heart in a dedicated folder.
5. Copy the R script (Supplementary File 1) onto the RStudio platform and run an R-based automated algorithm to assess MI. The algorithm computes planimetric infarct sizes for each slice and calculates the total infarct volume for the entire heart.
6. Export the dataset to an Excel file and analyze the data as needed.
7. Import all images of each heart into the ImageJ platform for subsequent 3D reconstruction.
8. Stack all images in a coherent, sequential order using the pop-up menu of the ImageJ platform.
9. Create a 3D construction using the surface plot function.
10. Open the Volume Viewer window and adjust all parameters for 3D projection.
    NOTE: The methodology for immunohistochemistry⁹ and videos for conventional TTC staining^4,5 have been previously reported and are not included in this manuscript.

Results

The key steps involved in performing microscopic TTC staining are illustrated in Figure 1. After two rounds of TTC staining, a distinct punched pattern became visible on the surface of the heart. Serial slicing of the entire heart at 100-µm intervals yielded approximately 80 tissue sections, with the ligation site located around the 60^th slice. In each section, the deep red staining of viable cardiomyocytes (viaCM) was clearly distinguishable from ...

Discussion

Myocardial infarction (MI), resulting from prolonged myocardial ischemia, remains a major global health concern, as ischemic injury often impairs the heart's contractile function required for effective blood circulation. Over the past several decades, significant efforts have been devoted to identifying interventions or compounds that enhance cardiac resistance to ischemic stress¹. Although alternative surrogate endpoints, such as enzyme release¹¹, post-ischemic functional ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.25x objective	Olympus	PlanApo 1.25X/0.04	Imaging
10 mL Syringers	B.Braum	4617100V	Retro-perfusion
2,3,5-triphenyltetrazolium chloride	Milipore	1.08380.0100	Chemicals
20 G cannula	BD	305895	Retro-perfusion
3  mL Syringers	B.Braum	4617022V	Retro-perfusion
3-way stopcock	B.Braum	16494C	Retro-perfusion
Aquamount	BDH	362262H	Cyrosection
Balance	Ohaus	N/A	Weighing chemicals
Binocular miccroscope	Leica	MZ6	Canulation
C57BL/6J	Janvier	N/A	Animals
Camera	Olympus	UC30	Imaging
Cover slips	Epredia	BB02400500A113MN20	Cyrosection
Cryostat	Leica	M850	Cyrosection
Double Distilled Water	MIlipore	N/A	Disolving chemicals
Fine dissecting scissors	AESCULAP	BC702R	Surgical Instruments
Fine micro-dissecting forceps	AESCULAP	FM001R	Surgical Instruments
Glass microscope slides	Engelbrecht	11102	Cyrosection
ImageJ	NIH	1.54g	Imaging processing
Isoflurane	Piramal	14150097146754	Chemicals
Isopropanol	Roth	0733.1	Chemicals
Light microscope	Olympus	BX61	Imaging
Micro-dissecting forceps	F.S.T	11152-10	Surgical Instruments
OCT compound	Sakura	4583	Chemicals
Petri dish	Therno Scientific	172931	Retro-perfusion
Phosphate Buffered Saline	Roth	9143.1	Chemicals
Rstudio	Posit Software	2024.09.0	Imaging processing
Silk suture	F.S.T	18020-60	Retro-perfusion
Software	Olympus	CellSens V3.2	Imaging
Stainless dissecting scissors	AESCULAP	BC257R	Surgical Instruments
Zamboni solution	Morphisto	12773.0050	Chemicals