Current Protocols

Manual analysis of skeletal muscle cross-sections is time-consuming and subject to error and user bias. To overcome these limitations, we developed and validated a semi-automated, quantitative, and reproducible image-analysis pipeline specifically tailored to quantify Pax7+ satellite cells, myonuclei, and cross-sectional area by fiber type. The workflow combines FIJI/ImageJ-based image preprocessing with CellProfiler, Cellpose, and a custom Python script to process and analyze immunohistological images of muscle tissue cross-sections. Outcomes include Pax7+ satellite cells and myonuclei quantified per fiber by fiber type, along with cross-sectional area, perimeter, and fiber type classification. This semi-automated approach provides a robust and efficient platform for high-throughput analysis of muscle tissue cross-sections from large datasets. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/729866v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@a3401dorg.highwire.dtl.DTLVardef@1c63145org.highwire.dtl.DTLVardef@ccbf76org.highwire.dtl.DTLVardef@2e0da0_HPS_FORMAT_FIGEXP M_FIG C_FIG

The bacterial three-hybrid (B3H) assay is a powerful genetic tool for detecting interactions between RNA and RNA-binding proteins (RBPs) and assessing the consequences of RBP mutations. This transcription-based system connects the strength of an RNA-protein interaction to the expression of a lacZ reporter gene in Escherichia coli cells. This in vivo approach allows researchers to dissect RNA-protein interactions within a cellular environment, bypassing the need for biochemical purification of RNAs or proteins. This chapter details a three-day protocol for generating quantitative B3H data. Since a significant challenge in B3H assays is RNA misfolding, we describe a recently optimized set of B3H constructs that mitigates this issue by isolating bait RNAs as discrete folding units.

Three-dimensional (3D) spheroid models have become essential in cancer biology, drug screening, and tissue engineering. However, their small size, fragile structure, and tendency to disintegrate during routine histoprocessing present persistent technical challenges. Conventional paraffin embedding often results in tissue fragmentation, loss of spatial orientation, and poor section quality, whereas cryosectioning often compromises cellular morphology. Here, we present a robust, cost-effective protocol for preserving and sectioning fragile 3D spheroids, resulting in high-quality histological sections with intact architecture and excellent cellular detail. The method involves optimized handling and embedding procedures that stabilize spheroids during standard formalin fixation, paraffin infiltration, and microtomy, eliminating mechanical distortion and preserving spherical integrity for consistent sectioning. We demonstrate successful application across different cell line spheroids, with subsequent compatibility with hematoxylin and eosin (H&E) staining protocols. Compared to conventional methods, our approach significantly reduces sample loss, improves inter-section reproducibility, and preserves fine structural features such as necrotic cores, proliferative zones, and extracellular matrix components. This protocol provides a reliable, accessible solution for routine histological analysis of fragile 3D spheroids, facilitating more accurate morphological and molecular assessment in translational research settings. Key featuresO_LIMaintains spheroid integrity: Prevents mechanical distortion, fragmentation, and loss of spatial orientation during processing. C_LIO_LISignificantly reduces sample loss: Decreases failure rate compared to traditional methods, conserving valuable samples. C_LIO_LIBroad spheroid compatibility: Works effectively with primary tumor-derived, stem cell-derived, and co-culture spheroid models. C_LIO_LIEnables high-quality sectioning and staining: Delivers consistent, reproducible sections that are fully compatible with H&E, IHC, and IF. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/743094v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1670c4org.highwire.dtl.DTLVardef@145810aorg.highwire.dtl.DTLVardef@1accb1org.highwire.dtl.DTLVardef@17481c0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Quantitative analysis of rodent behaviour is fundamental to neuroscience, preclinical drug discovery and neurotoxicology research. Although several commercial and open-source software packages are available for behavioural assessment, some are expensive, some require programming expertise, and some provide limited flexibility for user-defined experimental configurations. To address these limitations, we developed Robean, a freely available standalone software platform for automated rodent neurobehavioural analysis from both live camera feeds and pre-recorded videos. Robean provides an intuitive graphical user interface that enables users to design experimental arenas, define custom analysis zones, perform spatial calibration, and automatically track rodent movement without requiring programming knowledge. The software currently supports automated analysis of three widely used behavioural paradigms: the Morris Water Maze, Elevated Plus Maze, and Open Field Test. Robean extracts behavioural metrics including escape latency, path efficiency, platform crossings, target quadrant preference, thigmotaxis, locomotor activity, zone occupancy, arm entries, and centre exploration specific to behavioural tests. In addition, the software generates trajectory maps, occupancy heatmaps, comma-separated value (CSV) datasets, comprehensive PDF reports, and batch study summaries for multiple experimental sessions. Developed using open-source software technologies and distributed as a standalone freeware application, Robean provides an accessible and reproducible solution for behavioural neuroscience laboratories. Its modular architecture facilitates future integration of additional behavioural paradigms and analytical modules, making it a flexible platform for automated rodent behavioural assessment.

This protocol describes the workflow for the preparation of tissue samples for proteome and phosphoproteome analyses using mass spectrometry. The tissue samples are cryogenically pulverized and homogenized in a sucrose-based buffer to ensure proper tissue disruption. For depletion of lipid contaminants, proteins are purified using chloroform-methanol precipitation, followed by a resuspension in a urea-based buffer for enzymatic digestion. Peptides are desalted and enriched for phosphopeptides prior LC-MS/MS analysis. The workflow was developed for skin biopsies but is compatible with a broad range of tissue types. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=51 SRC="FIGDIR/small/732915v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@6a44aforg.highwire.dtl.DTLVardef@c34dc9org.highwire.dtl.DTLVardef@27d7ecorg.highwire.dtl.DTLVardef@1d1038e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

Metabolomics enables the profiling of small-molecule metabolites and thereby captures the biochemical state of a living organism at a given moment and enables to monitor its cellular responses to stimuli. This technique has become a powerful tool in pharmaceutical research, the food industry, and microbial research. Metabolomics aims to obtain an unbiased metabolic profile; however, this is complicated by compound instability, complex and often extensive sample processing, and nonlinear responses in mass spectrometry. Therefore, correcting for metabolite loss and mass spectrometry-related artifacts is essential, typically achieved through relative quantification against an isotopically labelled internal standard for each metabolite of interest. This article describes how to produce 13C-labelled yeast extract and its use as internal standard for metabolomics. More specifically, it provides step-by-step protocols for the fed-batch fermentation, quenching, metabolite extraction, and LC-MS and GC-MS characterization of the internal standard. It also includes a protocol explaining how to use the internal standard for the quantification of metabolites in yeast samples.

Advances in camera technology and computer vision techniques have allowed researchers to track animals in 3D in ways which previously were difficult or impossible. Many such 3D tracking tools make use of multiple cameras, but unfamiliarity with the principles and technology involved can make it difficult to employ such techniques. In this protocol, we describe Braid, open-source software for live, multi-camera 3D tracking of insects. Using background-subtraction, Braid performs detection of objects without requiring the use of physical markers affixed to the insect. Braid constructs low-latency 3D position estimates using Kalman filtering and nearest neighbor data association. We document in detail the process of tracking freely flying bees within a flight arena using Braid. This protocol includes instructions on installation, configuration of cameras, setup, calibration, and operation. Within the system described here, we demonstrate that Braid can achieve position estimates accurate to <1 millimeter (within a 0.3 cubic meter volume). These factors make Braid suitable for tracking small, fast-flying animals like insects. Braid's low latency allows live tracking, removing the necessity to collect large video files and making it suitable for integration in closed loop systems such as virtual reality. Code is available at https://github.com/strawlab/strand-braid

This step-by-step protocol provides a cross-species, non-surgical approach that enables prenatal gene delivery to the developing nervous system in rats and marmosets. Under transabdominal ultrasound guidance, intracerebroventricular injection of recombinant adeno-associated virus vectors into the fetal brain achieves robust and long-term transduction from prenatal stages into adulthood. This approach can be adapted to other species and target sites outside nervous system, enabling safe and selective intrauterine manipulation and the generation of diverse experimental models for basic and preclinical research. For complete details on the use and execution of this protocol, please refer to Ribeiro Gomes et al (2026)1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/737050v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@696364org.highwire.dtl.DTLVardef@fc3c7forg.highwire.dtl.DTLVardef@1e7c7caorg.highwire.dtl.DTLVardef@1edcef0_HPS_FORMAT_FIGEXP M_FIG C_FIG Before you beginExperimental procedures during gestation allow researchers to study developmental processes, including how manipulations of the fetus and its intrauterine environment influence biological outcomes. Ultrasound imaging guidance greatly facilitates such interventions by providing safe and targeted access to fetal compartments, including for prenatal gene delivery to developing neural cell populations. Critically, delivery of recombinant adeno-associated viruses (rAAVs) into the cerebrospinal fluid (CSF) of developing animals enables widespread gene transfer across the brain. The efficiency and distribution of transduction are strongly influenced by developmental stage, making the timing of delivery an important experimental variable. In altricial species such as mice, major developmental processes, including cortical lamination and the establishment of long-range connections, begin prenatally but continue throughout early postnatal life. In primates, however, development is more advanced at birth, and many equivalent developmental events are shifted to the prenatal period. Consequently, developmental stages that can be targeted postnatally in mice require prenatal access in primates. Here, we present a step-by-step protocol for ultrasound-guided fetal intracerebroventricular viral injection (FIVI) of rAAV in marmosets (Callithrix jacchus) and rats (Rattus norvegicus). The procedure was initially developed and optimized in rats before being translated to marmosets, small New World primates that share key developmental, anatomical, and functional characteristics with humans. Together, these models illustrate the cross-species applicability of the approach, while providing gene delivery strategies for both a genetically tractable rodent model and a translationally relevant nonhuman primate. FIVI enables broad gene transfer and stable, long-term transgene expression in wild type animals, facilitating the generation of complementary quasi-transgenic models for research and translational applications from prenatal development through adulthood.

While previous in vitro porcine (Sus scrofa) oviduct spheroid models often rely on manual selection of the formed aggregates, here, we establish a scalable and reproducible system for generating oviduct spheroids. We describe steps for isolating primary secretory and ciliated oviduct epithelial cells and their assembling them into spheroids by forced aggregation using the AggreWell platform. This enables the mass production of uniformly sized spheroids ready for use in a range of downstream applications, such as sperm-oviduct co-incubation experiments. For complete details on the use and execution of this protocol, please refer to Molina et al.1 Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/731731v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@20f94aorg.highwire.dtl.DTLVardef@ce80eeorg.highwire.dtl.DTLVardef@172c631org.highwire.dtl.DTLVardef@31f420_HPS_FORMAT_FIGEXP M_FIG C_FIG Before you beginThis protocol describes the isolation of primary porcine oviduct epithelial cells and their use in generating a three-dimensional (3D) spheroid model in vitro. The protocol is specifically designed to enrich and preserve ciliated oviduct epithelial cells, which are critical for physiologically relevant sperm-oviduct interaction studies yet are frequently underrepresented in conventional in vitro oviduct models. To minimize the de-differentiation of ciliated cells during culture, a specialized Incubation Medium is employed to maintain epithelial cell viability and ciliary function. The relatively brief spheroid formation period (48 h) further supports preservation of ciliary structure and activity prior to downstream functional applications. This protocol consists of four major steps. Steps 1 and 2 cover oviductal epithelial cell isolation through mechanical and enzymatic digestion, step 3 describes the spheroid formation and culture conditions, and step 4 applies the spheroid model to sperm co-incubations or immunofluorescent staining. Although optimized for the porcine isthmus, this protocol may also be suitable for the cells from the ampulla and adaptable to other large animal species.

The in vitro scratch wound healing assay is one of the most widely used methods for studying collective cell migration, but converting assay images into reproducible measurements remains a practical bottleneck of manual tracing, local software installation, parameter bookkeeping, and limited visibility into how the wound region was segmented. We present Cytomove, a browser-local software tool for reviewable scratch wound healing assay quantification. Cytomove imports local microscopy images, segments the wound region with an explainable variance-and-threshold pipeline implemented in client-side JavaScript without external image-processing dependencies, displays the segmentation as an inspectable overlay before any number is exported, supports single-image and grouped time-course analysis, and exports wound area, wound area fraction, wound width profile statistics, quality-control labels, and full analysis metadata as CSV, Excel, PNG, and ZIP. All processing runs in the browser or in a desktop package built on the same code; microscopy images never leave the users machine. In a preliminary comparison with the ImageJ/Fiji Wound Healing Size Tool (WHST) across five image sets and 31 paired measurements, Cytomove reproduced wound-area behaviour closely in a clean brightfield comparator sequence (mean absolute percentage error 4.1%, Pearson r = 0.9975) and in a phase-contrast time course approaching closure (median area error 6.6%, r = 0.9984), while surfacing near-closure and real-world acquisition difficulties through overlays and quality-control labels. Informal local testing indicates that typical single-image analysis completes within seconds in a modern browser, with no installation or dependency step. Cytomove lowers installation friction, keeps assay data local, and links every exported number to the segmentation image and parameters that produced it.

1. Video-tracking programs have now become an essential tool for researchers measuring animal behavior across biological fields. The panel of available programs is growing rapidly, providing researchers with numerous specific tools that will match their precise needs. However, their proliferation may complicate post-tracking data processing, and some programs do not even provide tools for correcting tracking errors or analysing tracking data. In the case of commercial software, the loss of access to a program due to budget limitations or researchers' mobility from one institution to another could prevent them from accessing and visualizing their tracking data. 2. There is therefore a growing need for an accessible and flexible tool to handle post-tracking processes such as the correction and analysis of tracking data obtained across different video-tracking programs. 3. We present here the latest update of the video tracking and analysis program AnimalTA. With this new release, we propose to solve the above-mentioned problems by providing the scientific community with a program that will allow for data importation from other video-tracking programs. Like in its previous versions, AnimalTA remains a free, open-source, and highly user-friendly program, ensuring that it will always be accessible without restriction. Now, with this new importation option, users who performed their tracking with other programs can benefit from AnimalTA's complete toolset of data visualization, correction, and analysis. 4. Finally, this article gives an overview of the other main improvements associated with this new release. The program is now faster in both video importation and tracking, proposes an amplified toolset for data visualisation and correction, and features new options for data analysis.

Derived Cleaved Amplified Polymorphic Sequences (dCAPS) assays have been widely performed historically to detect known base substitutions in many model organisms--notably Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, and Schizosaccharomyces pombe-- where chemical mutagens that induce point mutations are frequently used. With the rise of whole-genome sequencing and genome editing technologies, dCAPS is increasingly applied to detect diverse nucleotide changes in additional organisms, including Drosophila, zebrafish, mammals, and agricultural crops (e.g., Oryza sativa and Hordeum vulgare). However, a key limitation of dCAPS is that genomic target sites amenable to primer designs that both preserve PCR amplification and create recognition sites for inexpensive, high-performance restriction enzymes are scarce. Here we report One-step dual-primer dCAPS (OddCAPS), a modification that uses three primers in one reaction to overcome this constraint. Two of these primers, an intermediate primer and a dCAPS primer, sequentially introduce 1-2 base substitutions each into the amplicon, enabling up to four engineered base changes near the nucleotide of interest. By using the intermediate primer at 1/10-1/100 the concentration of the other primers, the desired product is generated directly in a single-tube, one-step PCR. Increasing the number of engineered substitutions improves the chance of using a researchers preferred restriction enzyme. In principle, having eight common restriction enzymes (BamHI, EcoRI, NheI, SalI, BglII, ClaI, HindIII, and MluI) suffices to detect any single-nucleotide variant in any biological or synthetic DNA sequence with this approach.

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

We present a simple and efficient way to add cis dialdehydes, phosphoryl groups, or cis diols to the 3 prime end of oligonucleotides using periodate oxidized nucleotides (oNTPs) and terminal deoxynucleotidyl transferase (TdT). The 3 prime end cis dialdehyde-modified oligos are generated by incubating TdT with an oligo for several minutes followed by addition of a oNTP and incubated at 30 degrees C for 30 minutes to an hour. After allowing the addition of the cis dialdehydes, heating the reaction mixture at 90 to 95 degrees C for 10 minutes yields oligonucleotides with 3 prime phosphoryl groups. The 3 prime cis diol modified oligos are synthesized by starting with 3 prime cis diol nucleotides (HO-NTPs). The cis dialdehyde modified oligonucleotides and cis diols may then be used for a variety of investigations such as studying the interaction of proteins with the 3 prime end of DNA, and possibly RNA. As an example, we demonstrate the efficacy of using an oligonucleotide modified with oGMP at the 3 prime end as an affinity label for TdT and identified a peptide fragment that has been shown to contain two of three aspartate residues found to be in the TdT active site.

To explore adapting qPCR systems for end-point nucleic acid quantification using dyes such as SYTO-9, we quantified serial dilutions of DNA and RNA standards in the range of 0.75 - 200 ng/{micro}l on 384-well qPCR devices. SYTO-9 fluorescence was successfully measured using standard SYBR Green settings. Blank-subtracted relative SYTO-9 signal showed a logarithmic dependence on DNA/RNA concentration (R2 > 0.95). Measurements were highly stable with different incubation times, temperatures of up to 95{degrees}C, and photobleaching. The described approach is a valuable QC option for high-throughput DNA/RNA isolations and could be adapted to additional fluorometric assays beyond nucleic acids.

Planning husbandry tasks and experiments with Drosophila melanogaster requires converting a target date into development times that depend on the rearing temperature. This calculation needs to be done for each cross, genotype, and temperature, and the risk of error grows quickly. Available laboratory management tools let users register stocks, crosses, and track them, but they do not create schedules based on a clear, adjustable thermal model. To fill that gap, we developed DrosoTracker, a self-contained web application that works offline and predicts Drosophila development with a thermal summation model recalibrated through regression on data from Powsner (1935) (T0 = 11.78 {degrees}C, DD = 116.38 {degrees}C{middle dot}days, R{superscript 2} = 0.997). The model offers an optional two-level calibration driven by user observations. A wild-type strain first adjusts the model to the laboratorys own conditions. Then each genotype is calibrated against that reference using a random-effects shrinkage estimator that accounts for measurement error and between-batch variability. The model creates schedules for husbandry tasks, evaluates adult cohort survival with the Kaplan-Meier estimator and the log-rank test, and calculates sample size for lifespan studies using Schoenfelds formula. The quantitative components were checked against independent references, including Rs survival package and manual calculations. Ongoing work is focused on validating the calibrated model using cohorts specifically bred for this purpose. DrosoTracker runs entirely in the browser, stores data locally, and is available in English and Spanish.

Background: Brain banks usually provide small tissue blocks fixed by immersion in neutral-buffered formalin (NBF). While still underexploited for research, gross anatomy laboratories could provide full brains fixed by perfusion with solutions better suited for gross anatomy dissection. However, the chemicals in these solutions might have a different impact on histology protocols for cell quantification than in NBF-fixed brains. The main goal of this study is to compare the effects on the number and size of labeled neurons of the primary motor cortex (PMC) of mouse brains fixed with three different solutions: (1) NBF, typical of brain banks, (2) a saturated salt solution (SSS), and (3) an alcohol-formaldehyde solution (AFS), both used in human anatomy laboratories. Methods: 27 C57BL/6J mouse brains were perfused with the NBF (N=9), SSS (N=9) or AFS (N=9), then cut in 40-m slices and processed with immunohistochemistry to target neurons. Various quantitative variables were assessed manually and automatically on photomicrographs of 3 regions of interest (ROIs) of the PMC per specimen, namely the total and individual neuronal profile areas, number and diameters. The effects of the three fixatives on these variables were compared using ANOVA or Kruskal-Wallis, depending on the distribution. For measures on individual cells, a generalized linear mixed model was applied. Dice coefficients and correlations were applied to evaluate the agreement of the manual and automatic methods. Results: There was no significant difference between the brains fixed by the three fixatives for the total and individual cell areas, the total cell count and the cell diameters. The values obtained from manual and automatic measures had an overall good agreement (Dice coefficients > 0.79). Conclusion: It was found that the SSS and AFS had similar impacts on the quantitative variables in the tissue as the NBF. These results are promising for neuroscientists interested in using brains from anatomy laboratories for quantitative research on neurons from the PMC.

Plasma membrane sheets generated by controlled mechanical disruption provide direct access to the cytosolic face of the plasma membrane while preserving the native organization of membrane-associated proteins and lipids. Here, we present a protocol for generating and validating sonication-derived plasma membrane sheets from cultured cells, primary neurons, and isolated synaptosomes. We further describe their application for live and fixed imaging of membrane protein localization, organization, conformational dynamics, and protein-protein interactions, as well as quantitative membrane-associated synaptic vesicle recruitment assays. This versatile platform preserves the native membrane environment while enabling direct visualization and quantitative analysis of membrane-associated processes at high spatial resolution. The protocol can be readily adapted to investigate diverse membrane proteins, lipid-dependent mechanisms, and vesicle tethering events across a wide range of cellular systems.

This protocol describes a standardized and reproducible minimally invasive approach for establishing mouse models of bisphosphonate-related osteonecrosis of the jaw (BRONJ) and osteoradionecrosis of the jaw (ORNJ). The method combines a unified low-trauma oral surgical procedure with disease-specific injury induction strategies to generate robust and clinically relevant models of jaw osteonecrosis. For BRONJ, systemic zoledronic acid administration is coupled with mandibular first molar extraction using tape-assisted mouth opening and customized bent micro-forceps, minimizing soft tissue damage and reducing procedural variability. For ORNJ, a customized lead-shielding platform enables precise, noninvasive mandible-targeted irradiation, producing reproducible bone injury while limiting off-target radiation exposure. Together, these complementary models provide a consistent and minimally invasive framework for investigating jaw osteonecrosis arising from distinct etiologies. The protocol supports comprehensive downstream analyses, including micro-computed tomography, histology, and immunofluorescence, and facilitates mechanistic studies of disease pathogenesis, bone regeneration, and therapeutic intervention.

Microcomputed tomography (micro-CT) is a useful tool that can be utilised for 3D structural characterisation and volumetric quantification of small biological specimens. Its potential application is particularly valuable within the field of cardiac development, where phenotypic profiling at the whole organ, cell, and molecular level is often most informative within the same sample. Consequently, this study sought to develop a multimodal imaging protocol to enable 3D phenotypic characterisation of embryonic avian hearts (iodine-based contrast X-ray imaging) prior to immunohistochemistry-based cell and molecular analysis. Micro-CT parameters were tested to establish an optimal protocol for 3D analysis of embryonic cardiac specimens across multiple developmental timepoints. Optimised parameters provided reliable and reproducible 3D analysis of cardiac macrostructures. Sodium thiosulphate treatment of X-ray imaged hearts effectively reversed the iodine-based contrast stain whilst maintaining antigen availability of nuclear, membranous, and cytoplasmic targets in traditional downstream imaging studies. Together, this study demonstrates a robust and highly efficient multimodal imaging strategy to comprehensively characterise cardiac morphology in avian embryos and may serve as a versatile foundation for a broad range of bioimaging applications within the wider scientific community.