Calcified Artery Preparation and Processing with Preserved Morphology and RNA for Digital Spatial Profiling

Spatial transcriptomics offers an advantage over both single-cell RNA seq and bulk RNA seq by preserving spatial information within the tissue. By using tools like RNAscope, computational deconvolution, and publicly available single-cell datasets, it is possible to map cellular behavior to its niche and gene profiles and build an integrated view of tissue biology. This technology's exploding.

Two May 2025 Nature"papers focus on spatial transcriptomics studies. If we chart PubMed, the curve is even steeper:roughly 150 spatial papers in 2023, nearly 300 in 2024, and on pace to surpass 500 publications in 2025. Spatial transcriptomics has already reshaped several major research areas, like cancer biology, neuroscience, immunology, and developmental biology.

Yet vascular biology is lagging. The heterogeneity and unique geometry of arteries and veins make them challenging for tissue attachment and RNA preservation. In addition, advanced vascular lesions feature calcification and thrombosis, which further reduce tissue integrity.

This protocol is specifically designed for vessel-like structures. We also include methodologies to reduce the cost and the necessary staining processes for assessing RNA, calcium deposition, and cells of interest. Given the current scarcity of vascular-focused spatial transcriptomics studies compared with other fields, this protocol is designed to be cost-efficient and beginner-friendly, providing vascular researchers with accessible tools to adopt spatial transcriptomics in their studies.

This protocol also is potentially helpful for large cohort samples for clinical experiments. In this protocol, we describe detailed procedures covering the careful handling of calcified artery tissues, property calcification methods aimed at preserving RNA quality and tissue morphology, staining techniques for verification and guidance during ROI selection, and final morphological marker staining on prepared slides, ensuring they are ready for processing on the digital spatial profiler instrument. Human tibial arteries, including the anterior tibial, posterior tibial, and perineal arteries, were harvested from patient amputation specimens and stored in DMEM containing 1%HEPES and 1%Pen-Strep.

Each artery is transferred to a 100-millimeter Petri dish with the media. Excess perivascular connective tissue is removed using sterile scissors and forceps, leaving only the arterial wall. The trimmed artery is transferred to a new Petri dish.

A sterile cutting board is prepared inside the dish. The arteries are cut into one-centimeter segments using a surgical blade. Two segments of each artery are snap-frozen in cryotubes in liquid nitrogen for cryopreservation and storage for future protein or RNA work.

The remaining segments are placed in 15-milliliter tubes containing 10%neutral buffered formalin and fixed at four degrees Celsius for 24 to 48 hours. The decalcification solution with 10%EDTA in an RNA stabilizing solution is prepared, adjusting the pH to 5.2 for the working solution. The fixed tissues are transferred to a new dish containing PBS, and the tissues are briefly washed.

Each sample is transferred to a 15-milliliter tube containing decalcification solution and agitated at four degrees Celsius for 72 hours. After decalcification is completed, the samples are washed twice using PBS with gentle agitation for five minutes. Tissues are transferred into cassettes.

The cassettes are placed into a tissue processor. Formalin processing is carried out for at least four hours. The time can be varied depending on the sample matrix.

Multiple artery segments are placed per block. The block is filled with paraffin and allowed to completely cool. Slides are prepared by marking the restricted tissue area on the slide backs.

Confirm that all segments fall within the restricted area required by the GeoMx DSP instrument. Excess paraffin is trimmed from the blocks. The blocks are then sectioned to five microns with low-profile blades.

The tissue ribbons are mounted within the marked area. At least four serial sections per block are mounted for different stains in the DSP experiment. Store slides at four degrees Celsius until use.

Next, RNAscope in-situ hybridization, as well as histological staining, is performed on serial prepared slides. In addition, another set of slides is stained with morphological markers, such as CD45 for immune cells and alpha-SMA for smooth muscle cells. The RNAscope in-situ hybridization procedure briefly consists of four major steps:slide baking as a pre-treatment, standard deparaffinization and rehydration, target retrieval, probe hybridization with signal amplification and dye staining for RNA visualization.

For histological validation, both H&E staining and Alizarin Red S staining are performed to allow precise localization of calcium deposits. Finally, morphological marker staining is performed with a standard immunohistochemistry protocol to identify the cells of interest, guiding the ROI selection. In this study, two EDTA-based protocols were compared:one, 10%EDTA alone for 72 hours, and two, 10%EDTA and RNA stabilizing solution for the same duration.

Morphology and RNA quality were assessed by RNAscope in-situ hybridization. Both methods preserved vessel architecture, but slides treated with the EDTA and RNA stabilizing solution cocktail displayed markedly stronger RNA signals, as demonstrated in the right panel, indicating that RNA stabilizing solution protects transcripts while EDTA chelates crystalline calcium phosphate and gradually softens the specimen without activating RNases. With the RNA preservation strategy optimized and quality confirmed, we demonstrate the steps for histologic staining for calcium deposition using hematoxylin and eosin and Alizarin Red S.Alizarin selectively binds ionic calcium salts and shifts in hue from orange to brick red, sensitively highlighting micro and macro calcifications, although it provides less cellular and extracellular matrix detail than H&E.

Using both stains maximizes structural and compositional information. After morphologic marker staining, slides are scanned on the GeoMx platform. Antibody CD45 marks immune cells, while alpha-smooth muscle actin labels vascular smooth muscle cells abundant in the media.

The selected ROIs encompass approximately 100 to 150 nuclei with adequate RNA yield. The ROIs were placed adjacent to and distant from calcified areas to compare spatial gene expression patterns relevant to tibial artery medial calcification. Despite some tissue loss during the multi-hour processing steps, multiple suitable ROIs were identified in each artery section.

To conclude, this workflow covers calcified artery tissue handling and decalcification through validation staining and ROI selection for a spatial transcriptomics study. In particular, combining H&E with calcium-specific Alizarin staining ensures precise localization of vascular calcification and maximizes both structural and compositional information for ROI placement. RNAscope analysis shows that decalcified arterial sections remain largely intact with sufficient RNA.