Protocol for quantitative image analysis of alizarin red staining to assess osteogenesis in cultured cells

Summary

Endothelial-to-mesenchymal transition (EndMT) generates mesenchymal-like cells with multilineage differentiation potential. We describe steps for inducing EndMT in human venous endothelial cells, directing their differentiation toward an osteogenic lineage, and assessing mineralization using an alizarin red assay. We then detail procedures for quantifying and classifying osteogenic capacity from alizarin red staining using custom image analysis pipelines. This protocol can also be used to quantify alizarin red staining in mouse-derived mesenchymal stem/stromal cells (MSCs) in vitro.

For complete details on the use and execution of this protocol, please refer to Kishta et al.¹

Graphical abstract

Highlights

• •Generation of mesenchymal-like cells from endothelial cells in culture
• •Osteogenic assay to assess mesenchymal cell osteogenic capacity in vitro
• •Alizarin red staining to quantify calcium deposition after osteogenic differentiation
• •Image-based computational quantification and classification of alizarin red staining

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Endothelial-to-mesenchymal transition (EndMT) generates mesenchymal-like cells with multilineage differentiation potential. We describe steps for inducing EndMT in human venous endothelial cells, directing their differentiation toward an osteogenic lineage, and assessing mineralization using an alizarin red assay. We then detail procedures for quantifying and classifying osteogenic capacity from alizarin red staining using custom image analysis pipelines. This protocol can also be used to quantify alizarin red staining in mouse-derived mesenchymal stem/stromal cells (MSCs) in vitro.

Before you begin

In cardiovascular disease, endothelial cells (ECs) can become activated and undergo EndMT,² a process associated with pathological vascular remodeling, including vascular stiffening and calcification.³ However, the extent and mechanism of EndMT contribution to these pathological outcomes remain incompletely understood.² Using an in vitro model,⁴ we found that EndMT-derived mesenchymal cells exhibit osteogenic differentiation upon stimulation. Although the differentiation of ECs into osteogenic cells through EndMT remains relatively understudied, several reports have described this cellular trajectory.^5,6

This protocol describes the steps required to generate mesenchymal progenitor cells from human umbilical vein ECs (HUVECs) through treatment with a combination of transforming growth factor-β2 (TGF-β2) and interleukin-1β (IL-1β) for 7 days, followed by osteogenic stimulation and quantitative assessment of mineralization using Alizarin Red staining and custom-built analysis pipelines.¹ This protocol builds on a previously established method of assessing osteogenesis⁷ and has also been successfully applied to mesenchymal cells derived from other tissues and species. The first section of this protocol (“in vitro system of EndMT”) describes the preparatory steps required to generate mesenchymal-like cells prior to osteogenic differentiation.

Innovation

This protocol establishes a quantitative imaging-based framework for the analysis and classification of mineralization using Alizarin Red-stained cells. While Alizarin Red staining is typically measured using spectrophotometric methods, our approach allows for direct visualization and quantitative analysis at the whole-well level. We developed customized computational image analysis pipelines that enable unbiased quantification and classification of Alizarin Red staining intensity across a continuous range of light to dark. A key advancement of this approach is that it provides a scalable, reproducible and quantitative readout suitable for high throughput studies.

In vitro system of EndMT (if necessary)

Timing: 13–14 days

Human umbilical vein endothelial cells (HUVECs) are cultured under optimal conditions in liquid medium to induce EndMT.

• 1.
  Passage HUVECs to P4.

  Note: The following steps are for HUVECs cryopreserved at passage P2-P3. Timing may vary depending on the passage of the frozen cells.

  • a.
    Thaw 1 mL of frozen HUVECs and transfer the cell suspension to 9–10 mL of complete EGM-2 medium in a T75 flask.
  • b.
    Incubate cells overnight (12–15 h) at 37°C, 5% CO₂.
  • c.
    The following day, carefully replace the medium with 10 mL of fresh EGM-2 to remove residual DMSO.

    Note: Centrifugation and resuspension are avoided to minimize cellular stress. By thawing cells late in the day and refreshing medium early the next day, baseline conditions are preserved, ensuring no additional stress influences EndMT induction.

  • d.
    When confluent, passage cells at a 1:4 ratio.

    • i.
      Remove medium from culture flask by gentle aspiration.
    • ii.
      Wash cells once with 10 mL of sterile 1× PBS.
    • iii.
      Remove the PBS, add 3 mL trypsin-EDTA to completely cover the cells and incubate at 37°C for 4–5 min.

      Alternatives: TrypLE 1× from Gibco™ (Cat. # 10718463) may also be used for enzymatic digestion of the cell monolayer.

    • iv.
      Confirm cell detachment under the microscope before proceeding.
    • v.
      Neutralize trypsin-EDTA by adding 7 mL of complete EGM-2 medium.
    • vi.
      Collect the 10 mL cell suspension in a sterile 15 mL tube.
    • vii.
      Centrifuge the cell suspension for 5 min at 161 × g at room temperature (RT; 20°C–25°C).
    • viii.
      Carefully aspirate the supernatant and resuspend cells in 4 mL fresh complete EGM-2 medium.
    • ix.
      Split cells at a 1:4 ratio by transferring 1 mL of cell suspension to a new T75 flask containing 9 mL of fresh EGM-2 medium.
• 2.
  Plate HUVECs for EndMT at P5.

  Note: Prepare sufficient wells for untreated (control) and EndMT-treated HUVECs, including wells designated for RNA extraction to confirm EndMT induction.

  CRITICAL: In addition, include an empty well (no cells) that can serve as a background reference for flatfield correction during brightfield imaging.

  CRITICAL: Wells designated for RNA extraction must be maintained in separate plates from those intended for osteogenic differentiation.

  • a.
    Once confluent, split HUVECs as described through steps 1a–1d (up to vii).
  • b.
    Pre-coat 6 well plates with 1 mL collagen (diluted 1:5 in PBS) per well and incubate at 37°C while splitting cells.
  • c.
    Determine cell concentration using an automated cell counter. Mix 20 μL of cell suspension with 20 μL Trypan Blue (1:1 v/v) to determine number of viable cells.
  • d.
    Remove the collagen solution from the 6-well plates.
  • e.
    Wash each well with 1 mL 1× PBS to remove residual collagen.
  • f.
    Aspirate the PBS and seed 70,000 live HUVECs per well in 1 mL of complete EGM-2 medium.
• 3.
  Start EndMT treatment 24 h after seeding HUVECs.

  • a.
    Prepare a master mix of EndMT induction medium by supplementing EGM-2 medium with TGF-β2 to a final concentration of 10 ng/mL and IL-1β to a final concentration of 1 ng/mL.

    CRITICAL: EndMT induction medium must be prepared fresh for each use and should not be stored for future use.

  • b.
    At 24 h post-seeding, replace the culture medium with EndMT induction medium for EndMT-treated HUVECs or with fresh complete EGM-2 medium for control wells.

    Note: This first medium change marks Day 0 of EndMT induction.

  • c.
    Refresh EndMT or control medium every 24 h for 7 days.
  • d.
    On Day 7 of EndMT, capture representative phase-contrast images of control and EndMT-treated HUVECs to assess morphological changes (Figure 1).

    Note: Images were captured using a Nikon Eclipse TS100 inverted microscope equipped with a Nikon Plan 10×/0.25 WD 10.5 objective lens and a Nikon DS-Fi2-L3 digital camera system. Images were acquired using the Tripolis 100 display system, maintaining the same acquisition settings across experimental conditions.

    Alternatives: Equivalent tissue culture microscopes may also be used.

  • e.
    For wells designated for RNA extraction:

    • i.
      Remove culture medium and wash once with 1 mL 1× PBS per well.
    • ii.
      Remove the PBS under the chemical fume hood.
    • iii.
      Add 700 μL of QIAzol lysis buffer per well and incubate at RT (20°C–25°C) for 5 min.
    • iv.
      Collect lysates in 1.5 mL tubes and proceed with RNA extraction, as described by Kishta et al. (2026),¹ to confirm EndMT induction.

Figure 1.

Phase-contrast images of control and EndMT-treated HUVECs on Day 7 of EndMT induction

Images were acquired using a Nikon Eclipse TS100 Microscope. Scale bars: 100 μm.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
EGM-2 BulletKit™	Lonza	Cat. # CC-4176
Fetal bovine serum (FBS)	Life Technologies	Cat. # 16140071
Phosphate buffered saline (PBS)	Gibco	Cat. # 10010023
Penicillin-streptomycin (P/S)	Gibco	Cat. # 15140122
Trypsin-EDTA	Gibco	Cat. # 25200056
Rat-tail collagen	Sigma-Aldrich	Cat. # C3867-1VL
Trypan Blue solution 0.4%	Sigma-Aldrich	Cat. # T8154
Human transforming growth factor β2 (TGF-β2)	PeproTech	Cat. # 100-35B
Human interleukin 1β (IL-1β)	PeproTech	Cat. # 200-01B
QIAzol Lysis Reagent	Qiagen	Cat. # 79306
Paraformaldehyde (PFA)	VWR International	Cat. # J61899
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	Cat. # D2653-5X5ML
Dexamethasone	Sigma-Aldrich	Cat. # D4902
L-ascorbic acid-2-phosphate (L-AA)	Sigma-Aldrich	Cat. # A8960
β-Glycerophosphate disodium pentahydrate (β-GP)	Calbiochem	Cat. # 35675
Gibco Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax	Gibco	Cat. # 61965026
Alizarin Red S	Sigma-Aldrich	Cat. # A5533-25G
Hydrochloric acid	Sigma-Aldrich	Cat. # 258148
Ammonium hydroxide	Sigma-Aldrich	Cat. # 338818
MilliQ H20	Sartorius	Arium Comfort
Experimental models: Cell lines
Human umbilical vein endothelial cells	Lonza	Cat. # CC-2519; RRID: CVCL_2959
Mouse derived mesenchymal stromal cells from vein and aorta	This study	N/A
Software and algorithms
Zen 2.3 Pro	Zeiss	https://www.zeiss.com
Fiji/ImageJ	FIJI	https://imagej.net/software/fiji
Stained wells analysis.ijm	This study	N/A
BioRender	BioRender	https://www.biorender.com
Other
1.5 mL tubes	Eppendorf	Cat. # 15625367
6-well plates	Corning	Cat. # CLS353224
Greiner polypropylene tubes 50 mL	Merck	Cat. # 227270
Greiner polypropylene tubes 15 mL	Merck	Cat. # 188261-N
0.22 μm filters	Merck	Cat. # SLGPM33RS
T75 flasks	Corning	Cat. # CLS430720U
Pipette tips	Sarstedt	Cat. # 70.3010.275 Cat. # 70.3030.265 Cat. # 70.1189.215 Cat. # 70.3050.255
Brightfield microscope	Zeiss	Axio Observer Z1
Phase contrast microscope	Nikon	Eclipse TS100
Microscope color camera	Zeiss	AxioCam MR R3
Objective5×/0.15	Zeiss	Plan-Neofluar
Digital camera system	Nikon	DS-Fi2-L3
Automated cell counter	BioRad	TC20
Incubator	PHCbi	IncuSafe CO2
Centrifuge	Fisher Scientific	GT1
Bio-Safety cabinet	MAT	BioMAT2
Water bath	Grant	Sub Aqua Pro

Materials and equipment

EndMT inducing medium

Reagent	Stock concentration	Final concentration	Amount
EGM-2 BulletKit™ with Penicillin-streptomycin (P/S)	N/A	N/A	10 mL
Human transforming growth factor β2 (TGF-β2)	20 μg/mL	10 ng/mL	5 μL
Human interleukin 1β (IL-1β)	10 μg/mL	1 ng/mL	1 μL
Total	N/A	N/A	10 mL

Prepare fresh for each use.

Osteogenic differentiation medium

Reagent	Stock concentration	Final concentration	Amount
Fetal bovine serum (FBS)	N/A	10%	10 mL
Penicillin-streptomycin (P/S)	N/A	1%	1 mL
L-ascorbic acid-2-phosphate (L-AA)	5 mM	50 mM	1 mL
β-Glycerophosphate disodium pentahydrate (β-GP)	1 M in DMEM	10 mM	1 mL
Dexamethasone	1 mM in DMSO	100 nM	10 μL
Gibco Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax	N/A	N/A	87 mL
Total	N/A	N/A	100 mL

Store at 4°C, use within 1 month.

Alizarin Red staining solution

Reagent	Final concentration	Amount
Alizarin Red S	0.02 g/mL	2 g
MilliQ Water	N/A	100 mL
Total	N/A	100 mL

Store at room temperature (20°C–25°C), protect from light, use within 1 month.

Step-by-step method details

Osteogenesis assay

Timing: 19–21 days

This section describes the induction of osteogenic differentiation in mesenchymal-like cells in culture. In this protocol, osteogenic differentiation is demonstrated using mesenchymal-like cells derived from ECs via EndMT and is subsequently validated in mouse aortic and venous stromal mesenchymal cells (MSCs).

• 1.
  Prepare solutions as described in the materials and equipment section.

  • a.
    Prepare osteogenic medium in a 100 mL flask.
  • b.
    Mix thoroughly and filter the medium using a 0.22 μm filter.

    CRITICAL: Store medium at 4°C for no longer than 1 month.

  • c.
    Prepare the Alizarin Red staining solution in a medium Schott flask.
  • d.
    Adjust the pH to 4.1–4.3 using HCl or NH₄OH, as required, and filter using a 0.22 μm filter.

    CRITICAL: Store Alizarin Red solution at RT (20°C–25°C) for up to 1 month, protected from light.

• 2.
  Cell differentiation assay.

  • a.
    On Day 7 of EndMT, replace the culture medium with osteogenic medium in both control and EndMT-treated HUVECs.

    Note: This first osteogenic medium change marks Day 0 of osteogenesis.

    CRITICAL: Trypsinization, cell counting, and reseeding into 24-well plates at a density of 40,000 cells per well were tested prior to osteogenic differentiation, as has been previously described in MSCs.⁷ Although reseeded cells attached successfully, osteogenic differentiation was impaired and followed by loss of cell viability. Consequently, all osteogenic differentiation experiments were performed without cell trypsinization or reseeding.

  • b.
    Incubate cells at 37°C and 5% CO₂ for 19–21 days.

    Note: A range of 19–21 days was defined for osteogenic differentiation to accommodate variability.

  • c.
    Refresh medium every third day for the duration of the differentiation assay.
• 3.
  Alizarin Red staining.

  • a.
    On the assay’s endpoint, discard the osteogenic medium.
  • b.
    Gently wash cells once with 1 mL 1× PBS to remove residual medium.
  • c.
    Fix cells with 1 mL 4% paraformaldehyde (PFA) per well for 10 min at RT (20°C–25°C).
  • d.
    Remove the PFA and wash cells twice with 1 mL per well Milli-Q water.
  • e.
    Add 500 μL of Alizarin Red solution to each well and incubate for 15 min at RT (20°C–25°C) in the dark.
  • f.
    Carefully remove the Alizarin Red solution by tilting the plate and aspirating with a P200 pipette.
  • g.
    Wash each well twice with 1 mL per well of Milli-Q water.

    CRITICAL: Add the water gently without touching the bottom of the well to avoid disturbing the stained mineralized matrix (Figure 2).

  • h.
    Perform a third wash with 1 mL of Milli-Q water and do not discard.
  • i.
    Image whole wells with a brightfield microscope.

    Note: Osteogenic differentiation of these cells was independently validated by supplementary approaches including osteogenic gene expression analysis by qPCR.¹

• 4.
  Brightfield image acquisition.

  • a.
    Acquire tiled brightfield images of each well.

    Note: Images were acquired using a Zeiss Axio Observer Z1 microscope equipped with a 5×/0.15 Plan-Neofluar objective, an AxioCam MR R3 color camera, and Zeiss Zen 2.3 Pro software. Illumination and exposure were set to 10% light and 15 ms and kept constant across all samples. Images were acquired as 12-bit RGB (Red, Green, and Blue) images, resulting in histogram maxima of approximately 2200 (maximum possible value 4096).

    CRITICAL: Image approximately 75% of each well to avoid well walls and meniscus-induced shadows

  • b.
    Acquire a flatfield image by imaging an out of focus empty well (no cells).

    Note: This flatfield image is used as reference image for flatfield correction during stitching.

  • c.
    Stitch tiled images using Zeiss Zen 2.3 software:

    • i.
      Open the Processing tab.
    • ii.
      Under Methods, select the Stitching option.
    • iii.
      Under Method Parameters, open the New Output tab and select Fuse Tiles.
    • iv.
      Click Apply to generate a fused image from the fused tiled images (Figure 3).
    • v.
      Create a new folder and save the fused image as a.czi file.

      Note: Use the folder containing the fused.czi files as the input directory when running the Fiji/ImageJ macro.

  • d.
    Open the fused.czi images in Fiji/ImageJ using the same settings for all wells for qualitative analysis of Alizarin Red staining.

Figure 2.

Alizarin Red-positive cells following osteogenic differentiation

Representative image of a 6-well plate following Alizarin Red staining of untreated (top row) and EndMT-treated HUVECs (bottom row) after 19–21 days of osteogenic differentiation. EndMT was induced for 7 days prior to osteogenic differentiation.

Figure 3.

Whole-well Alizarin Red staining of EndMT-treated cells

Representative well containing EndMT-treated HUVECs stained with Alizarin Red, showing unstitched tiled acquisition (left) and the stitched whole-well image (right) used for downstream image analysis. Yellow arrows show representative areas of tile boundaries. Scale bars: 2000 μm.

Quantification and classification of alizarin red staining

Timing: Dependent on the number of images to be analysed

• 5.
  Perform whole-well image analysis using custom-built macros.

  • a.
    Quantitative method:

    • i.
      Download the macro file named “Methods S1”. In the video tutorial, the macro is named “Stained wells analysis.ijm”.
    • ii.
      Create an output folder to save the results generated by the macro.
    • iii.
      Open the input folder containing the stitched.czi images exported from Zen 2.3.
    • iv.
      Import the stitched images into Fiji/ImageJ and run the macro according to the accompanying video tutorial (Methods Video S1).
    • v.
      Quantify the total area positive for Alizarin Red staining.
    • vi.
      Calculate the proportion of Alizarin Red-positive area relative to total well surface area.

      Note: The macro automatically thresholds and measures stained and unstained regions and saves the output values in the designated output folder (Figure 4).

    • vii.
      Determine the unstained area by subtracting the total Alizarin Red-positive area from the total well surface area.
  • b.
    Semi-quantitative method:

    • i.
      Apply predefined thresholds to classify Alizarin Red-positive areas into light, medium, and dark staining categories (Figures 5 and 6).

      Note: Threshold values should be determined based on background signal and staining intensity distribution. To establish thresholds, select representative regions of the RGB image containing each staining category. Next, convert images from RGB to HSB (Hue, Saturation, and Brightness) color space in Fiji/ImageJ and record saturation values from the saturation histograms (Figure 5). Once these values are standardized, keep them constant throughout the experiment.

• 6.
  Macro adaptation (Optional).

  CRITICAL: Adapt the macro when using a microscope or image acquisition system different to the one described in this protocol.

  • a.
    Modify the file extension in line 42 of the macro to match the input image format. The default setting is.czi.
  • b.
    Confirm that the correct image series is selected in line 43. Select Series 3 for pyramidal.czi files, as demonstrated in the video tutorial (Methods Video S1).
  • c.
    Perform image down-sampling before analysis if the input file is not pyramidal.
  • d.
    Adjust the values in the “makeOval” command in line 54 to define the area of interest and crop the image. This removes shadows caused by the meniscus and bright white corners.
  • e.
    Standardize the array values defined in line 77 according to the specific camera used.

Figure 4.

Image analysis pipeline

Whole-well images were acquired using a 5× objective alongside a flatfield image. Images were stitched and flatfield corrected in ZEN to remove microscope-related shadowing. The resulting images were imported into Fiji/ImageJ, where Alizarin Red staining was segmented into 3 categories based on saturation in HSB color space. The surface area occupied by each staining category was measured and calculated against the surface of the whole well. A color-coded map where each stain category has its own color (blue for unstained, green for light, yellow for medium, red for dark), was created alongside the original image as quality control. As an output, the macro delivers a table of results containing absolute surface area measurements and a color-coded image for each well quantified.

Figure 5.

Determination of saturation thresholds for Alizarin Red staining

Saturation histograms of small regions containing homogenous staining were analysed in HSB color space to define the values used in the macro. Unstained regions showed a distinct peak between saturation values 0 and 64, whereas light staining extended up to 164. Medium staining values concentrated between 164 and 232, while dark staining occurred between 232 and 255. The light-stained region is surrounded by unstained areas, accounting for the additional peak at lower saturation values (left peak). Similarly, the medium-stained region contains a small number of light and dark areas, resulting in broader distribution. Across a whole-well image, saturation values are distributed across the full histogram.

Figure 6.

Whole-well Alizarin Red staining quantification of EndMT-treated cells

Representative whole-well image of EndMT-treated HUVECs cultured in osteogenic differentiation medium and stained with Alizarin Red (left), with the corresponding colormap showing classified staining intensities (right). Scale bars: 2000 μm.

Application of alizarin red quantification in mouse osteogenic cells in culture

We next validated the use of custom-built macros in mouse osteogenic stromal cells. Mouse aorta-derived or vein-derived mesenchymal stromal cells were cultured in osteogenic conditions for 21 days (20,000 cells/cm²), stained for Alizarin Red, then full wells imaged and quantified as described above (steps 1–5) (Figure 7).

Figure 7.

Whole-well Alizarin Red staining quantification of murine mesenchymal stromal cells

Representative whole-well images of Alizarin Red-stained MSCs derived from mouse aorta (upper panel) and vein (lower panel), with corresponding quantification color maps. Scale bars: 2000 μm.

Expected outcomes

The described model of EndMT (TGF-β2 + IL-1β co-treatment for 7 days) should cause HUVECs to acquire an elongated and larger cell morphology (Figure 1) accompanied by changes in molecular signature such as decreased expression of endothelial genes (e.g., Pecam1) and increased expression of mesenchymal genes (e.g., Tagln), as described.¹ It is not necessary to use EC-derived mesenchymal cells and one can directly use MSCs as previously described.² If using HUVECs to acquire mesenchymal-like cells through EndMT, RNA yield from one well (from a 6-well plate) at Day 7 of EndMT induction is expected to be anywhere from 70–150 ng/μL. Gene expression analysis for EndMT confirmation will result in reduction of Pecam1 by 30%–50%, while Tagln expression may increase up to 60-fold.¹ Similar findings are expected when measuring protein changes.

Following the completion of osteogenic differentiation and Alizarin Red staining, wells should appear red (Figure 2). It is possible that staining intensity may vary across wells and/or experimental groups. In cases where washing is not done carefully, the cell layer can lift (Figure 2, well #6). Following successful staining and imaging using the Zen software, the user will have.czi images that will need to be converted to.tiff and opened on Fiji/ImageJ. After running the macrocode, the user will have an Excel file with raw values of intensity, the analysed images as shown in Figures 6 and 7, and a Log.txt file detailing the details of the run.

Limitations

The in vitro model of EndMT used in this protocol may not fully recapitulate the mineralization process occurring in vivo. In addition, image analysis depends on the consistent staining quality and image acquisition across experiments. While the workflow enables whole-well quantification and classification of Alizarin Red staining, direct comparison with conventional dye extraction-based spectrophotometric measurement was not performed. Instead, osteogenic differentiation was validated using supplementary approaches including osteogenic gene expression analysis by qPCR and RNA sequencing.¹

Troubleshooting

Problem 1

EndMT induction is not successful.

Potential solution

EndMT treatment must be done every 24 h using freshly prepared EndMT induction medium with freshly thawed cytokines. It is recommended to aliquot the cytokines to volumes that ensure fresh aliquots may be used daily.

Problem 2

Cells detach during the differentiation protocol.

Potential solution

Replacing media should be done carefully by removing and adding media on the walls of the wells so no pressure is added directly to the cell layer which can detach upon differentiation.

Problem 3

At the end of Alizarin Red staining, red-stained cells detach and float in the well.

Potential solution

To prevent loss of stained cells, all solutions used (PBS, Milli-Q water, Alizarin Red, and PFA) should be added and aspirated by using a P200 pipette, with the pipette tip positioned against the wall of each well. This technique avoids direct pressure on the cells and prevents contact with the cell layer.

Problem 4

Shadows from the well wall are quantified as dark red Alizarin Red staining.

Potential solution

Image 75% of the well to avoid these artifacts.

Problem 5

Issues with running the macro on Fiji/ImageJ.

Potential solution

Ensure user specific changes have been made as described on the video tutorial and notes within the protocol text.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Mihaela Crisan (mihaela.crisan@ed.ac.uk).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contacts, Franceska Kishta (fkishta@ed.ac.uk), Tanushree Tripathi (tanushree.t98@gmail.com), and Matthieu Vermeren (matthieu.vermeren@ed.ac.uk).

Materials availability

This study did not generate any new unique reagents.

Data and code availability

• •All data presented are derived from the same experiments as reported in Kishta et al.¹ The associated original code is attached in the paper.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

We acknowledge the British Heart Foundation (BHF) PhD Studentship Grant (FS/4yPhD/F/20/34126) to F.K.; the Institutional Strategic Support Fund (ISSF3) pump-priming grant (J22739) and BHF Centre for Research Excellence (REA3) pump-priming grant (R45929) to M.C; and the British Heart Foundation Chair of Translational Sciences (CH/11/2/28733), BHF Program Grant (RG/F/24/110149), and the European Research Council funded the UK Guarantee (APP43780) to A.H.B. The graphical abstract was created using Biorender.com. The figures were created using Affinity Designer v2.

Author contributions

F.K., T.T., A.H.B., and M.C. conceptualized, designed the experiments, performed the research, and analyzed and/or interpreted data. M.V. helped with images, designed and wrote the macrocode, applied the macrocode to stained images, and analyzed the data. F.K. wrote the original draft. All authors edited, reviewed, and commented on the manuscript.

Declaration of interests

The authors declare no competing interests.