Method Article

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

DOI:

10.3791/69159

⸱

January 23rd, 2026

 ,  ,  , 
1Division of Vascular Surgery, Department of Surgery, University of Washington, 2Department of Laboratory Medicine and Pathology, University of Washington, 3Division of Vascular Surgery, VA Puget Sound Health Care System

* These authors contributed equally

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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'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.

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Protocol

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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

  1. Tissue trimming
    1. 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's Modified Eagle's Medium (DMEM) media containing 1% of 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid (HEPES) and 1% penicillin-streptomycin (P/S).
    2. Place the harvested anterior tibial (AT), posterior tibial (PT), and peroneal tibial (PR) arteries (ideally > 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.
    3. 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.
    4. Briefly rinse the trimmed artery with Dulbecco'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.
    5. Cut the artery cross-sectionally into 1 cm segments with a surgical blade.
  2. Tissue fixation and storage
    1. Snap-freeze two segments in cryotubes for cryopreservation in liquid nitrogen (LN2) and store them for future protein/RNA work.
    2. Place the remaining segments in 15 mL tubes containing 10% NBF and fix at 4 °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.

2. Decalcification, tissue processing, and sectioning

  1. 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.
  2. Briefly rinse the fixed tissues in PBS manually. Transfer each sample to a 15 mL tube containing the decalcification solution and agitate at 4 °C for 72 h.
  3. When decalcification is complete, wash the samples in PBS (2 × 5 min). Place tissues into cassettes and load them into a tissue processor using a 4 h processing program.
  4. 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.
  5. Trim excess paraffin. Section the blocks at 5 µm 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.
  6. Prepare at least four serial sections per block for different stains and the digital spatial profiling (DSP) experiment. Store slides at 4 °C until use (ideally < 6 months).

3. RNAscope In-situ hybridization (ISH) and histological staining

  1. RNAscope ISH
    1. Bake slides in a dry oven at 60 °C overnight to enhance tissue adhesion to the slides.
    2. Deparaffinization: Submerge slides in xylene (3 × 5 min), followed by 100% ethanol (2 × 3 min). Dry slides at 60 °C for 7 min.
    3. Perform the RNAscope 2.5 HD Red ISH assay according to the manufacturer's protocol. Block endogenous peroxidase with 3% H2O2 for 10 min at RT; rinse in distilled (DI) water.
    4. Incubate slides in pre-heated, 100 °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 °C for 7 min. Create a hydrophobic barrier for solutions around the tissue using a hydrophobic barrier PAP pen.
    5. Place the slides in a hybridization oven rack, add 200 µL of digestive enzyme supplement (prediluted by the vendor in dropper bottles) to each slide, and incubate at 40 °C for 15 min. Rinse the slides twice in DI water.
    6. Apply positive control probe Human Ppib to the slides and incubate for 2 h at 40 °C. Wash the slides 2x in wash buffer.
    7. Perform sequential amplification: AMP1 at 40 °C for 30 min; AMP2 at 40 °C for 15 min; AMP3 at 40 °C for 30 min; AMP4 at 40 °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.
    8. 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.
    9. Counterstain with Gill'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.
  2. Alizarin Red S staining
    1. For additional serial sections, repeat deparaffinization and rehydration in Steps 3.1.2.
    2. Confirm the Alizarin Red S working solution pH is 4.1-4.3.
    3. Immerse slides in Alizarin Red S for 30-60 s, or until calcium deposits develop an orange-red color.
    4. Shake off excessive staining dye. Dehydrate with 20 quick dips in 100% acetone, then 20 dips in acetone:xylene (1:1).
    5. Clear the slides in two fresh baths of xylene, 3 min each. Coverslip with mounting medium.

4. GeoMx DSP slide preparation and ROI selection

  1. 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.
  2. Bake slides in a dry oven at 60 °C overnight to enhance tissue adhesion to the slides.
  3. Deparaffinization and rehydration: Submerge slides in xylene (3 × 5 min), followed by 100% ethanol (2 × 5 min), and 95% ethanol diluted in DEPC-treated water for 5 min. Transfer the slides to 1× PBS for 1 min.
  4. Incubate slides in pre-heated, 100 °C Tris/EDTA pH 9.0 antigen retrieval buffer for 20 min, and transfer the slides to 1× PBS for 5 min at RT.
  5. Incubate slides in preheated 1.0 µg/ml Proteinase K for 15 min and rinse in 1× PBS for 5 min at RT.
  6. Postfix the slides in 10% NBF (200 µL per slide) for 5 min at RT, and wash in PBS for 5 min.
  7. 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.
  8. Add 200 µL 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 °C overnight.
  9. Dip slides in saline-sodium citrate buffer (SSC, 2×), and allow the hybridization slips to slide off by themselves within 5 min.
  10. Wash the slides in stringent wash buffer composed of equal parts 4× SSC and deionized formamide at 37 °C, 25 min per wash. Wash the slides in 2× SSC buffer, 2 min per wash.
  11. Block the slides with Buffer W at RT for 30 min.
  12. Cover the tissue in primary antibody solution (Rabbit anti-CD45) for 1 h in the humidity chamber slide box at RT, wash with 2× SSC buffer 3x.
  13. Apply secondary antibody solution (Goat anti-Rabbit IgG-AF647) for 1 h and wash in 2× SSC buffer.
  14. Cover tissue in labeled antibody (Mouse anti-SMA-AF488) for 1 h in the humidity chamber slide box at RT. Wash in 2× SSC buffer.
  15. Stain the slides for 10 min in 2 µM Syto83 nuclear marker diluted in Buffer W for 10 min.
  16. 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's layer-specific location.
    NOTE: Exercise caution to prevent air-bubble formation when applying the hybridization slip to the tissue sections.

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Results

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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...

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Discussion

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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...

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Disclosures

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The authors have no conflicts of interest to declare.

Acknowledgements

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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.  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.  We would like to acknowledge the imaging service provided by the Histology and Imaging Core (HIC) at the University of Washington.  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:  UW Spatial Biology Core.  We would also like to acknowledge Nishi Ivanov at the Diabetes Research Center at the University of Washington for histological staining.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Dulbecco's Modified Eagle's Medium (DMEM), high glucose, pyruvate Thermo Fisher Scientific11995065
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, 1 MThermo Fisher Scientific15630080
Penicillin-Streptomycin (P/S), 10,000 U/mLThermo Fisher Scientific15140122
10% Neutral Buffered Formalin (NBF)Millipore Sigma65346-M
Ethylene-diamine-tetraacetic acid (EDTA)Millipore Sigma3620
RNAlater Stabilization SolutionThermo Fisher ScientificAM7024RNA stabilization solution (RSS)
Hydrogen peroxide (H2O2)Advanced Cell Diagnostics, Inc322335
Target retrieval solutionAdvanced Cell Diagnostics, Inc322001
Protease PlusAdvanced Cell Diagnostics, Inc322331digestive enzyme supplement
DapB (negative control)Advanced Cell Diagnostics, Inc310043
RNAscope wash buffer (RNAscope 2.5 HD Red ISH assay kit)Advanced Cell Diagnostics, Inc320058
AMP1Advanced Cell Diagnostics, Inc324511
AMP2Advanced Cell Diagnostics, Inc324512
AMP3Advanced Cell Diagnostics, Inc324513
AMP4Advanced Cell Diagnostics, Inc324514
AMP5 RedAdvanced Cell Diagnostics, Inc324515
AMP6 RedAdvanced Cell Diagnostics, Inc324516
Fast Red AAdvanced Cell Diagnostics, Inc324517
Human PpibAdvanced Cell Diagnostics, Inc313901
Gill’s hematoxylinMillipore SigmaGHS132
Antigen retrieval, Tris/EDTA pH 9Thermo Fisher Scientific00-4956-58
Proteinase KThermo Fisher ScientificAM2546
Hybridization solution-Hs WTANanostring121401102
Hybrisliphybridization slip
Hybridization solution-Buffer RNanostring121300313
Rabbit anti-CD45 Cell Signaling1317
Buffer WNanostring121300313
Goat anti-Rabbit IgG-AF647Jackson Immunoresearch111-605-144
Mouse anti-SMA-AF488 Thermo Fisher Scientific53-9760
Syto83 Thermo Fisher ScientificS11364
Surgical bladeBD Biosciences371110
Superfrost  Plus Microscope SlidesThermo Fisher Scientific1255015
ImmEdge pen
Microtome bladesThermo Fisher Scientific22-500-125
RNase AWAYRNase-inactivating solution
Equipment
HybEZ hybridization oven Advanced Cell Diagnostics, Inc321710
GeoMx Digital Spatial ProfilerNanostring Model: GeoMx DSP
Data access platform: GeoMx DSP Control Center (Version 3.1.2.12)		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.

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Tags

Spatial TranscriptomicsRNA PreservationTissue DecalcificationRNAscope HybridizationHistological StainingAlizarin Red StainingTissue MicroarrayRegion Of Interest

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