Name: Calcified Artery Preparation and Processing with Preserved Morphology and RNA for Digital Spatial Profiling (Video) | JoVE Journal
Uploaded: 2026-01-23T14:40:00.000+00:00
Duration: 9 min 12 s
Description: Learn calcified artery preparation for spatial transcriptomics using decalcification and RNA preservation techniques. This protocol enables precise digital spatial profiling of vascular tissue morphology and gene expression.

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5 minutes

This work was supported by a seed grant from the VA Puget Sound, VA Merit award I01 BX004975-02, and resources and the use of facilities at the VA Puget Sound Health Care Center.&#160; The work is that of the authors and does not necessarily reflect the position or policy of the Department of Veteran Affairs or the United States government.&#160; We would like to acknowledge the imaging service provided by the Histology and Imaging Core (HIC) at the University of Washington.&#160; We would like to thank Dr Shreeram Akilesh for his guidance on DSP experiments at the University of Washington and his group at MANTIS Labs:&#160; UW Spatial Biology Core.&#160; We would also like to acknowledge Nishi Ivanov at the Diabetes Research Center at the University of Washington for histological staining.

All procedures for collecting human arterial specimens were approved by the University of Washington Human Subjects Division Institutional Review Board (IRB) and the IRB of the VA Puget Sound Health Care System (Seattle, WA). Written informed consents were obtained from the patients before tissue collection. The materials and equipment used for this study are listed in the Table of Materials.
1. Tissue trimming and fixing
<ol>
	<li>Tissue trimming
	<ol>
		<li>Prepare the transportation bag using multi-layered packaging, containing an inner tube, a secondary container, an outer shipping canister, and a biohazard specimen bag. Fill the inner 50 mL centrifuge tube with 30 mL of storage Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) media containing 1% of 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid (HEPES) and 1% penicillin-streptomycin (P/S).</li>
		<li>Place the harvested anterior tibial (AT), posterior tibial (PT), and peroneal tibial (PR) arteries (ideally &#62; 5 cm in length) separately in the 50 mL centrifuge tubes. Transfer the canisters on ice from the operating room to the tissue culture room. 
		NOTE: It is recommended that the harvested sample length be at least 5 cm to allow for replicates and sufficient tissue to be cryostored for other assessments or validation.</li>
		<li>Transfer each artery to a 100 mm Petri dish with the media. Remove excess perivascular connective tissues using sterile scissors and forceps, leaving only the arterial wall (adventitia inward). 
		NOTE: Handle and trim the tissues in a biosafety cabinet wiped with an RNase inactivation solution to prevent RNA contamination. Use sterile scalpels and forceps; between samples, rinse them in sterile and nuclease-free water to remove residues and wipe thoroughly with 70% ethanol.</li>
		<li>Briefly rinse the trimmed artery with Dulbecco&#39;s Phosphate Buffered Saline (PBS) in a new Petri dish. Set a sterile cutting board inside a fresh dish and move the artery onto it.</li>
		<li>Cut the artery cross-sectionally into 1 cm segments with a surgical blade.</li>
	</ol>
	</li>
	<li>Tissue fixation and storage
	<ol>
		<li>Snap-freeze two segments in cryotubes for cryopreservation in liquid nitrogen (LN2) and store them for future protein/RNA work.</li>
		<li>Place the remaining segments in 15 mL tubes containing 10% NBF and fix at 4 &#176;C for 24-48 h for 1 cm arterial segments. 
		NOTE: Use of 10% NBF is recommended. If using 4% paraformaldehyde (PFA), prepare it freshly or verify a neutral pH before use to minimize RNA damage.</li>
	</ol>
	</li>
</ol>
2. Decalcification, tissue processing, and sectioning
<ol>
	<li>Prepare the decalcification solution: dissolve solid ethylene-diamine-tetraacetic acid (EDTA) to 10% (w/v) in RNA stabilization solution (RSS). Filtration is not required. Adjust the pH to 9.2 with sodium hydroxide (NaOH) pellets to fully dissolve the EDTA, then lower to 5.2 with 10 M HCl, which is the typical working pH of RSS and helps preserve RNA integrity.</li>
	<li>Briefly rinse the fixed tissues in PBS manually. Transfer each sample to a 15 mL tube containing the decalcification solution and agitate at 4 &#176;C for 72 h.</li>
	<li>When decalcification is complete, wash the samples in PBS (2 &#215; 5 min). Place tissues into cassettes and load them into a tissue processor using a 4 h processing program.</li>
	<li>Embed multiple artery segments per tissue block. Mark the restricted tissue area on the slide backs and verify that all sections fall within this area.</li>
	<li>Trim excess paraffin. Section the blocks at 5 &#181;m with a microtome and low-profile blades, and mount the tissue sections within the marked slide area. 
	NOTE: Discard the first few sections after facing and collect subsequent tissue sections. Use RNase-free brushes and clean the water bath between batches of samples.</li>
	<li>Prepare at least four serial sections per block for different stains and the digital spatial profiling (DSP) experiment. Store slides at 4 &#176;C until use (ideally &#60; 6 months).</li>
</ol>
3. RNAscope In-situ hybridization (ISH) and histological staining
<ol>
	<li>RNAscope ISH
	<ol>
		<li>Bake slides in a dry oven at 60 &#176;C overnight to enhance tissue adhesion to the slides.</li>
		<li>Deparaffinization: Submerge slides in xylene (3 &#215; 5 min), followed by 100% ethanol (2 &#215; 3 min). Dry slides at 60 &#176;C for 7 min.</li>
		<li>Perform the RNAscope 2.5 HD Red ISH assay according to the manufacturer&#39;s protocol. Block endogenous peroxidase with 3% H2O2 for 10 min at RT; rinse in distilled (DI) water.</li>
		<li>Incubate slides in pre-heated, 100 &#176;C retrieval buffer (diluted 1:10 in DI water) for 15 min (use 200 mL of retrieval buffer for a rack of up to 24 slides in a steamer), dip in 100% ethanol 5 times, and dry slides at 60 &#176;C for 7 min. Create a hydrophobic barrier for solutions around the tissue using a hydrophobic barrier PAP pen.</li>
		<li>Place the slides in a hybridization oven rack, add 200 &#181;L of digestive enzyme supplement (prediluted by the vendor in dropper bottles) to each slide, and incubate at 40 &#176;C for 15 min. Rinse the slides twice in DI water.</li>
		<li>Apply positive control probe Human Ppib to the slides and incubate for 2 h at 40 &#176;C. Wash the slides 2x in wash buffer.</li>
		<li>Perform sequential amplification: AMP1 at 40 &#176;C for 30 min; AMP2 at 40 &#176;C for 15 min; AMP3 at 40 &#176;C for 30 min; AMP4 at 40 &#176;C for 15 min; AMP5 at RT for 30 min; and AMP6 at RT for 15 min. Wash for 2 x 2 min in between.</li>
		<li>Add Fast Red working solution, incubate at RT for 10 min. Drain, submerge slides in DI water, then dip 5 times in fresh DI water. Confirm that the Ppib positive-control spots appear as discrete, punctate red dots.</li>
		<li>Counterstain with Gill&#39;s 1 hematoxylin, dip 5 times in 2 changes of DI water, blue in 0.02% ammonium hydroxide, and dip 5 times in 2 changes of DI water. Confirm that nuclei stain blue and tissue morphology is preserved. Allow slides to dry overnight and coverslip with mounting medium.</li>
	</ol>
	</li>
	<li>Alizarin Red S staining
	<ol>
		<li>For additional serial sections, repeat deparaffinization and rehydration in Steps 3.1.2.</li>
		<li>Confirm the Alizarin Red S working solution pH is 4.1-4.3.</li>
		<li>Immerse slides in Alizarin Red S for 30-60 s, or until calcium deposits develop an orange-red color.</li>
		<li>Shake off excessive staining dye. Dehydrate with 20 quick dips in 100% acetone, then 20 dips in acetone:xylene (1:1).</li>
		<li>Clear the slides in two fresh baths of xylene, 3 min each. Coverslip with mounting medium.</li>
	</ol>
	</li>
</ol>
4. GeoMx DSP slide preparation and ROI selection
<ol>
	<li>Before starting, clean all equipment and containers with RNAse-inactivating solution and dry or rinse with DEPC-treated water to prevent RNA contamination. Follow the instructions for the spatial RNA profiling assay.</li>
	<li>Bake slides in a dry oven at 60 &#176;C overnight to enhance tissue adhesion to the slides.</li>
	<li>Deparaffinization and rehydration: Submerge slides in xylene (3 &#215; 5 min), followed by 100% ethanol (2 &#215; 5 min), and 95% ethanol diluted in DEPC-treated water for 5 min. Transfer the slides to 1&#215; PBS for 1 min.</li>
	<li>Incubate slides in pre-heated, 100 &#176;C Tris/EDTA pH 9.0 antigen retrieval buffer for 20 min, and transfer the slides to 1&#215; PBS for 5 min at RT.</li>
	<li>Incubate slides in preheated 1.0 &#181;g/ml Proteinase K for 15 min and rinse in 1&#215; PBS for 5 min at RT.</li>
	<li>Postfix the slides in 10% NBF (200 &#181;L per slide) for 5 min at RT, and wash in PBS for 5 min.</li>
	<li>Remove the slides from PBS, tap on a paper towel, wipe away excess liquid surrounding the tissue with a Kimwipe, and set in the hybridization staining rack.</li>
	<li>Add 200 &#181;L of hybridization solution (diluted 1:10 in Buffer W) to each slide and apply a hybridization slip. Close the hybridization chamber, insert it in the oven, and incubate at 37 &#176;C overnight.</li>
	<li>Dip slides in saline-sodium citrate buffer (SSC, 2&#215;), and allow the hybridization slips to slide off by themselves within 5 min.</li>
	<li>Wash the slides in stringent wash buffer composed of equal parts 4&#215; SSC and deionized formamide at 37 &#176;C, 25 min per wash. Wash the slides in 2&#215; SSC buffer, 2 min per wash.</li>
	<li>Block the slides with Buffer W at RT for 30 min.</li>
	<li>Cover the tissue in primary antibody solution (Rabbit anti-CD45) for 1 h in the humidity chamber slide box at RT, wash with 2&#215; SSC buffer 3x.</li>
	<li>Apply secondary antibody solution (Goat anti-Rabbit IgG-AF647) for 1 h and wash in 2&#215; SSC buffer.</li>
	<li>Cover tissue in labeled antibody (Mouse anti-SMA-AF488) for 1 h in the humidity chamber slide box at RT. Wash in 2&#215; SSC buffer.</li>
	<li>Stain the slides for 10 min in 2 &#181;M Syto83 nuclear marker diluted in Buffer W for 10 min.</li>
	<li>Scan the slides with RNA profiling instrument to visualize the morphological marker staining. Select ROIs proximal and distal to calcified lesions of each artery tissue, annotating each ROI&#39;s layer-specific location. 
	NOTE: Exercise caution to prevent air-bubble formation when applying the hybridization slip to the tissue sections.</li>
</ol>

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The authors have no conflicts of interest to declare.

This protocol details the steps required to prepare calcified human tibial arteries for spatial transcriptomic profiling on the NanoString GeoMx DSP. Tibial arteries, like all vessels, possess concentric intima, media, and adventitia layers, each harboring distinct cell populations that orchestrate injury and repair. Maintaining the architecture of these layers is essential for layer-resolved transcriptomic analyses, yet several often-overlooked preparatory steps can compromise that goal.
Afte...

Over the past decade, next-generation sequencing and high-resolution imaging have converged to advance spatial transcriptomics, a method of whole transcriptome quantification while preserving each transcript&#39;s precise location in tissue sections1,2. When paired with the rapidly maturing single-cell RNA-sequencing (scRNA-seq) field, spatial approaches provide an efficient route to link gene signatures with specific cell types, states, and niches within a tissue or organ, greatly enriching our understanding of disease mechanisms3,4,5,6,7. Multiple commercial platforms are being developed, such as 10X Visium, NanoString GeoMx and CosMx, Slide-seq, and DBiT-seq, each supported by protocols that are optimized for soft, highly cellular tissues such as tumors and brain tissue. The field is experiencing explosive growth, with PubMed indexing approximately 150 publications in 2023, nearly 300 in 2024, and on pace to surpass 500 in 2025. Despite this surge, vascular biology remains underrepresented compared with cancer, neuroscience, and immunology.
Hard or mineralized tissues, such as bone, cartilage, teeth, and plaque-laden, calcified arteries or veins, have proved far less amenable. The technical hurdles are multifaceted. First, the tissues with dense mineral matrices demand chelation or acid-based removal, aggressive treatments that may fragment RNA and distort tissue morphology. Second, diseased tissues, either injured or fibrotic, often contain few living cells embedded in copious extracellular matrix (ECM), reducing transcript yield and complicating data normalization. In addition, vascular tissues possess a thin, concentric architecture (intima, media, adventitia) wrapped around a hollow lumen. Limited wall thickness, especially the single-cell-layer intima, means capture areas may hold scant tissues, and sections can tear or detach during multi-hour staining and hybridization steps. As a result, spatial transcriptomic studies of vascular tissue remain scarce, even though cardiovascular diseases consistently rank among the leading causes of death and serious morbidity worldwide. Protocol papers that do exist overwhelmingly emphasize data processing, visualization, or platforms other than NanoString GeoMx8,9,10,11,12,13. Among these papers, few describe bench-level methods for processing diseased tibial arteries, a clinically relevant vessel in peripheral artery disease (PAD), especially for diabetic patients who make up an increasing proportion of the affected population.
To address this gap, we present a comprehensive workflow for calcified human tibial arteries obtained from PAD patients who underwent amputation. The protocol spans: (1) tissue trimming and dissection immediately after operating-room retrieval; (2) proper tissue fixation and decalcification that maintain integrity of tissue morphology and RNA; (3) construction of cost-saving tissue microarrays (TMAs) to minimize batch effects; (4) RNAscope in situ hybridization and histological calcium staining for quality control; and (5) Region of interest (ROI) selection strategy specifically designed for calcified tibial arteries on the GeoMx Digital Spatial Profiler (DSP) platform. These steps include all procedures prior to sample collection on the DSP instrument, library preparation, sequencing, and data analysis.
Although demonstrated on the referenced DSP platform, the principles can guide tissue preparation for 10X Visium or other platforms, and the methodology is readily adaptable to additional vascular beds or animal models. This protocol may be adapted for biobank and post-mortem tissues, but its success depends critically on tissue preservation, fixation, and storage conditions. Tissues should be fixed immediately after harvest to minimize ischemic time (ideally within 1 h). Standard 10% neutral-buffered formalin (NBF) is recommended for at least 16 h at room temperature (RT). Tissues should be embedded promptly after fixation and should not be stored in ethanol for more than 3 days. Formalin-fixed paraffin-embedded (FFPE) blocks processed and stored under recommended conditions remain suitable for spatial profiling for approximately 3 years, although RNA quality declines with block age. Accordingly, RNA quality assessment is recommended for low-cellularity samples, blocks that are stored over 6 months, biobank specimens, and post-mortem tissues. The minimum acceptable RNA quality depends on the assay selected, ROI selection, desired resolution, and core facility recommendations. In practice, approximately 50-100 nuclei per segment are commonly recommended for robust results. The DSP platform supports both FFPE and fresh frozen tissues. In this protocol, we focus on FFPE tissues.
While this study focuses on spatial transcriptomics, it is readily integrated with scRNA-seq. Using published scRNA-seq datasets and computational spatial deconvolution, cell-type proportions can be estimated for each spatial spot, and scRNA-seq-defined cell states can be projected onto tissue architecture. By maintaining both structure and RNA integrity, our protocol enables downstream single-cell or single-molecule validation, maximizing biological insight from precious vascular specimens and wider adoption of spatial transcriptomics in vascular biology research.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM), high glucose, pyruvate&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11995065</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, 1 M</td><td>Thermo Fisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Penicillin-Streptomycin (P/S), 10,000 U/mL</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>10% Neutral Buffered Formalin (NBF)</td><td>Millipore Sigma</td><td>65346-M</td><td></td></tr><tr><td>Ethylene-diamine-tetraacetic acid (EDTA)</td><td>Millipore Sigma</td><td>3620</td><td></td></tr><tr><td>RNAlater Stabilization Solution</td><td>Thermo Fisher Scientific</td><td>AM7024</td><td>RNA stabilization solution (RSS)</td></tr><tr><td>Hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Advanced Cell Diagnostics, Inc</td><td>322335</td><td></td></tr><tr><td>Target retrieval solution</td><td>Advanced Cell Diagnostics, Inc</td><td>322001</td><td></td></tr><tr><td>Protease Plus</td><td>Advanced Cell Diagnostics, Inc</td><td>322331</td><td>digestive enzyme supplement</td></tr><tr><td>DapB (negative control)</td><td>Advanced Cell Diagnostics, Inc</td><td>310043</td><td></td></tr><tr><td>RNAscope wash buffer (RNAscope 2.5 HD Red ISH assay kit)</td><td>Advanced Cell Diagnostics, Inc</td><td>320058</td><td></td></tr><tr><td>AMP1</td><td>Advanced Cell Diagnostics, Inc</td><td>324511</td><td></td></tr><tr><td>AMP2</td><td>Advanced Cell Diagnostics, Inc</td><td>324512</td><td></td></tr><tr><td>AMP3</td><td>Advanced Cell Diagnostics, Inc</td><td>324513</td><td></td></tr><tr><td>AMP4</td><td>Advanced Cell Diagnostics, Inc</td><td>324514</td><td></td></tr><tr><td>AMP5 Red</td><td>Advanced Cell Diagnostics, Inc</td><td>324515</td><td></td></tr><tr><td>AMP6 Red</td><td>Advanced Cell Diagnostics, Inc</td><td>324516</td><td></td></tr><tr><td>Fast Red A</td><td>Advanced Cell Diagnostics, Inc</td><td>324517</td><td></td></tr><tr><td>Human Ppib</td><td>Advanced Cell Diagnostics, Inc</td><td>313901</td><td></td></tr><tr><td>Gill&amp;rsquo;s hematoxylin</td><td>Millipore Sigma</td><td>GHS132</td><td></td></tr><tr><td>Antigen retrieval, Tris/EDTA pH 9</td><td>Thermo Fisher Scientific</td><td>00-4956-58</td><td></td></tr><tr><td>Proteinase K</td><td>Thermo Fisher Scientific</td><td>AM2546</td><td></td></tr><tr><td>Hybridization solution-Hs WTA</td><td>Nanostring</td><td>121401102</td><td></td></tr><tr><td>Hybrislip</td><td></td><td></td><td>hybridization slip</td></tr><tr><td>Hybridization solution-Buffer R</td><td>Nanostring</td><td>121300313</td><td></td></tr><tr><td>Rabbit anti-CD45&amp;nbsp;</td><td>Cell Signaling</td><td>1317</td><td></td></tr><tr><td>Buffer W</td><td>Nanostring</td><td>121300313</td><td></td></tr><tr><td>Goat anti-Rabbit IgG-AF647</td><td>Jackson Immunoresearch</td><td>111-605-144</td><td></td></tr><tr><td>Mouse anti-SMA-AF488&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>53-9760</td><td></td></tr><tr><td>Syto83&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>S11364</td><td></td></tr><tr><td>Surgical blade</td><td>BD Biosciences</td><td>371110</td><td></td></tr><tr><td>Superfrost&amp;nbsp; Plus Microscope Slides</td><td>Thermo Fisher Scientific</td><td>1255015</td><td></td></tr><tr><td>ImmEdge pen</td><td></td><td></td><td></td></tr><tr><td>Microtome blades</td><td>Thermo Fisher Scientific</td><td>22-500-125</td><td></td></tr><tr><td>RNase AWAY</td><td></td><td></td><td>RNase-inactivating solution</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HybEZ hybridization oven&amp;nbsp;</td><td>Advanced Cell Diagnostics, Inc</td><td>321710</td><td></td></tr><tr><td>GeoMx Digital Spatial Profiler</td><td>Nanostring&amp;nbsp;</td><td>Model: GeoMx DSP&lt;br/&gt; Data access platform: GeoMx DSP Control Center (Version 3.1.2.12)</td><td>Channel scan setting: for FITC, explosure time is 300 ms, and fluorophore is AlexaFluro 488; for channel Cy3, exposure time is 50 ms, and fluorophore is Syto83; and for channel Cy5, exposure time is 300 ms, and fluorophore is AlexaFluor 647.</td></tr></tbody></table>

calcified artery preparation and processing with preserved morphology and rna for digital spatial profiling

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

Spatial transcriptomics maps whole-transcriptome profiles directly onto tissue sections, correlating between cellular neighborhoods and gene expression and how these activities drive health and disease. Although the approach has transformed oncology, neuroscience, immunology, developmental sciences, and many fields, it has been less widely used in vascular biology, even though vascular disease remains a leading cause of death worldwide. Two obstacles stand out: arteries and veins offer little flat surface for sectioning, and diseased specimens, often atherosclerotic, thrombosed, plaque-laden, or calcified, require harsh treatments such as decalcification that jeopardize morphology and RNA integrity. Yet these same vessels, with their concentric intima, media, and adventitia layered structures, are ideal for spatial analysis because each layer hosts distinct cell types and gene programs that interact across the wall.
Few detailed protocols address how to prepare vascular tissues for spatial transcriptomics. This shortage limits researchers' ability to exploit the technique's full potential. To bridge the gap, we present a step-by-step workflow optimized for human tibial arteries with advanced lesions. The protocol covers tissue handling, fixation, and proper decalcification that preserve structure and RNA quality, followed by histological staining to grade calcification severity. We also describe the construction of tissue microarray (TMA) to curb batch effects and region of interest (ROI) selection strategies on the NanoString GeoMx Digital Spatial Profiler (DSP). 
By lowering technical barriers, this protocol enables vascular researchers to generate reliable spatial transcriptomic data and study layer-specific transcriptional activities in healthy and diseased vessels. We anticipate that it will accelerate the discovery of mechanisms underlying calcification, inflammation, intimal atherosclerosis, and other vascular pathologies and foster broader adoption of spatial transcriptomics in vascular biology.

The protocol outlined here guides investigators from the receipt of tibial arteries to the generation of TMA slides that retain both morphology and RNA integrity for spatial transcriptomics analysis. It also details histological methods for visualizing calcium deposits in arteries, morphological marker staining, and ROI selection strategies.
In Figure 1, arteries are fixed in 10% NBF (step 2). NBF penetrates tissue rapidly, minimizing RNA degradation, and its phos...

This protocol includes a step-by-step workflow for calcified vascular specimens: tissue handling, decalcification, RNA validation, calcification level detection, and region-of-interest selection strategies on the Nanostring GeoMx Digital Spatial Profiler (DSP). The goal is to present a comprehensive method for preserving vascular tissue morphology and RNA for reliable spatial transcriptomic analysis.

Spatial transcriptomics maps whole-transcriptome profiles directly onto tissue sections, correlating between cellular neighborhoods and gene expression ...

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.

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

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