Mapping of heart tissue using Visium Spatial Gene Expression for FFPE

Natalia-Del Pilar Vanegas; Lorena Rosas; Ana L. Mora; Mauricio Rojas

Jun 04, 2026

Mapping of heart tissue using Visium Spatial Gene Expression for FFPE

DOI

https://dx.doi.org/10.17504/protocols.io.14egnpxbpv5d/v1

Mapping of heart tissue using Visium Spatial Gene Expression for FFPE

Natalia-Del Pilar Vanegas¹,
Lorena Rosas¹,
Ana L. Mora¹,
Mauricio Rojas¹

¹Division of Pulmonary, Critical Care, & Sleep Medicine, Department of Internal Medicine, The Ohio State University, Wexner Medical Center, Columbus, OH, 43210, USA.

TriState SenNet
Cellular Senescence Network (SenNet) Method Development Community

Lorena Rosas

The Ohio State University

DOI: https://dx.doi.org/10.17504/protocols.io.14egnpxbpv5d/v1

Protocol Citation: Natalia-Del Pilar Vanegas, Lorena Rosas, Ana L. Mora, Mauricio Rojas 2026. Mapping of heart tissue using Visium Spatial Gene Expression for FFPE. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnpxbpv5d/v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: June 04, 2026

Last Modified: June 04, 2026

Protocol Integer ID: 318551

Keywords: Heart spatial mapping, Visium FFPE, Cardiac transcriptomics, Spatial gene expression, 10x genomics visium spatial gene expression reagent kit, using visium spatial gene expression, precise mrna localization within intact cardiac tissue architecture, mapping of heart tissue, mapping gene expression, visium spatial gene expression, mapping gene expression in formalin, transcriptome probe hybridization, intact cardiac tissue architecture, cardiac biology, human heart tissue, precise mrna localization, heart tissue, studies of cardiac biology, ffpe

Funders Acknowledgements:

TriState SenNET (Lung and Heart) Tissue Map and Atlas consortium - NIH

Grant ID: U54AG075931

Abstract

We describe a validated workflow for spatially mapping gene expression in formalin-fixed paraffin-embedded (FFPE) human heart tissue using the 10x Genomics Visium Spatial Gene Expression Reagent Kit for FFPE (CG000407, Rev D). Whole-transcriptome probe hybridization enables precise mRNA localization within intact cardiac tissue architecture, supporting studies of cardiac biology, fibrosis, ischemia, and cardiomyopathy. This protocol complies with SenNet Consortium data submission requirements.

Guidelines

Tissue collection may only proceed with prior approval from the user’s Institutional Ethics Board (IEB) or an equivalent ethics committee.

Tissue quality assessment 
Steps
Store FFPE heart tissue blocks at 4°C. Limit storage to <10 years to preserve RNA integrity.
Cut three 10 µm sections into a 1.5 mL LoBind tube.
Extract RNA with the Qiagen RNeasy FFPE Kit (Cat. 73504) per the RNeasy FFPE Handbook. Assess DV200 on the Agilent 2100 Bioanalyzer
Proceed only if ALL criteria are met: DV200 ≥30%, no splits or artifacts, and regions of interest are present on the section.  GO/NO-GO: DV200 <30%: do not proceed — test an alternative block.
Perform H&E staining on an adjacent section to visualize cardiomyocytes, interstitial fibroblasts, and vascular structures for spatial data interpretation.
 

 Sample preparation

Sectioning (per CG000408 Rev D)
A
Trim the FFPE block. The tissue footprint must not exceed 6.5 × 6.5 mm to fit within the Visium capture area.
Section at 5 µm onto the Visium Spatial Gene Expression Slide, centered on the capture area.NOTE: Heart tissue is dense — ensure the microtome blade is sharp to minimize compression artifacts.
Float sections on a 42°C water bath; dry completely at 42°C for three hours and place in a desiccator and keep overnight at room temperature before proceeding.
 

Deparaffinization, H&E, Imaging & Decrosslinking (per CG000409 Rev D)
NOTE: Run all steps on a test slide first. Capture brightfield TIFF image before decrosslinking — required for Space Ranger alignment.
Steps
Deparaffinize: xylene or substitute → graded ethanol series → water                                          (per CG000409).
H&E stain: Hematoxylin → bluing → Eosin Y → dehydrate → mount.
Image at 20× brightfield; save as TIFF for Space Ranger.
Decrosslinking per CG000409 temperature and time specifications.
CRITICAL: Under- or over-decrosslinking reduces
probe hybridization efficiency. Optimize on test slide.
 

Library Construction (CG000407 Rev D)
Follow CG000407 precisely for all reagent volumes, incubation times, and thermocycler programs.

3.1 Probe Hybridization
Steps
Prepare probe hybridization mix (CG000407 Table 1). Keep on ice until use.
Apply to tissue capture area; cover with Visium Slide Seal. Hybridize overnight (16-24h) in a Thermal cycler with the following incubation protocol and start program.
NOTE: Hybridization efficiency is the primary determinant of library complexity.
 3.2 Probe Ligation
Steps
Wash slides to remove unbound probes               (per CG000407).
Apply ligation mix; incubate at 37°C for 1 h. Wash to remove unligated probes.
 
3.3 Probe Release & Extension
Steps
Apply RNase mix (incubate at 37°C for 30 min, CG000407 program); followed Permeabilization Mix (incubate at 37°C for 40 min, CG000407 program).
Apply Probe Extension Mix (incubate at 45°C for 25 min, CG000407 program).
Apply KOH Mix and transfer released probe
solution to a LoBind tube immediately.
CRITICAL: pH of KOH
 
3.4 PCRAmplification & Indexing
Steps
Determine cycle number by qPCR according to Step 4.1, CG000407.
PCR amplify with Dual Index adapters- TS Set A well ID. Incubate in a thermal cycler with CG000407 program based on cell cycle determined.
Perform final SPRI cleanup; elute in Buffer EB.
 

Library QC & Sequencing

QC MetricAcceptable RangeNotes
DV200  ≥30%Tissue pre-screen
  
Library concentration
    2–10 nMQubit dsDNA HS
Library size
    240 bp
  Bioanalyzer HS DNA chip
  
Reads per spot
    35,000
  Recommended
  
Sequencing Configuration
• Read 1: 28 cycles  |  Read 2S: 50 cycles  |  Index i7: 10 cycles  | Index i5: 10 cycles
• Platform: Illumina NovaSeq 6000, NextSeq 2000, or equivalent
• Minimum depth: 25,000 readsper tissue-covered spot

Data Processing
• Process FASTQs with Space Ranger (spaceranger count) using GRCh38 and the Visium FFPE probe set.
• Confirm spatial barcode–image alignment via Loupe Browser or Space Ranger HTML summary.
• Archive raw FASTQs and Space Ranger outputs 
   (filtered_feature_bc_matrix/, spatial/,    tissue_positions.csv).

Protocol references

10x Genomics (2022). Visium Spatial Gene Expression Reagent Kits for FFPE User Guide. Document No. CG000407, Rev E. 10x Genomics, Inc. https://www.10xgenomics.com/support/spatial-gene-expression ffpe/documentation/steps/library-construction/visium-spatial-gene-expression-reagent-kits-for-ffpe-user-guide

10x Genomics (2022). Visium Spatial Gene Expression for FFPE — Tissue Preparation Guide. Document No. CG000408, Rev D. 10x Genomics, Inc. https://www.10xgenomics.com/support/spatial-gene-expression-ffpe/documentation/steps/tissue-prep/visium-spatial-gene-expression-for-ffpe-tissue-preparation-guide

10x Genomics (2022). Visium Spatial Gene Expression for FFPE — Deparaffinization, H&E Staining, Imaging & Decrosslinking. Document No. CG000409, Rev D. 10x Genomics, Inc.    https://www.10xgenomics.com/support/spatial-gene-expression-ffpe/documentation/steps/tissue-staining

	Steps
	Store FFPE heart tissue blocks at 4°C. Limit storage to <10 years to preserve RNA integrity.
	Cut three 10 µm sections into a 1.5 mL LoBind tube. Extract RNA with the Qiagen RNeasy FFPE Kit (Cat. 73504) per the RNeasy FFPE Handbook. Assess DV200 on the Agilent 2100 Bioanalyzer
	Proceed only if ALL criteria are met: DV200 ≥30%, no splits or artifacts, and regions of interest are present on the section. GO/NO-GO: DV200 <30%: do not proceed — test an alternative block.
	Perform H&E staining on an adjacent section to visualize cardiomyocytes, interstitial fibroblasts, and vascular structures for spatial data interpretation.

	A
	Trim the FFPE block. The tissue footprint must not exceed 6.5 × 6.5 mm to fit within the Visium capture area.
	Section at 5 µm onto the Visium Spatial Gene Expression Slide, centered on the capture area.NOTE: Heart tissue is dense — ensure the microtome blade is sharp to minimize compression artifacts.
	Float sections on a 42°C water bath; dry completely at 42°C for three hours and place in a desiccator and keep overnight at room temperature before proceeding.

	Steps
	Deparaffinize: xylene or substitute → graded ethanol series → water (per CG000409).
	H&E stain: Hematoxylin → bluing → Eosin Y → dehydrate → mount.
	Image at 20× brightfield; save as TIFF for Space Ranger.
	Decrosslinking per CG000409 temperature and time specifications. CRITICAL: Under- or over-decrosslinking reduces probe hybridization efficiency. Optimize on test slide.

	Steps
	Prepare probe hybridization mix (CG000407 Table 1). Keep on ice until use.
	Apply to tissue capture area; cover with Visium Slide Seal. Hybridize overnight (16-24h) in a Thermal cycler with the following incubation protocol and start program. NOTE: Hybridization efficiency is the primary determinant of library complexity.

	Steps
	Wash slides to remove unbound probes (per CG000407).
	Apply ligation mix; incubate at 37°C for 1 h. Wash to remove unligated probes.

	Steps
	Apply RNase mix (incubate at 37°C for 30 min, CG000407 program); followed Permeabilization Mix (incubate at 37°C for 40 min, CG000407 program).
	Apply Probe Extension Mix (incubate at 45°C for 25 min, CG000407 program).
	Apply KOH Mix and transfer released probe solution to a LoBind tube immediately. CRITICAL: pH of KOH

	Steps
	Determine cycle number by qPCR according to Step 4.1, CG000407.
	PCR amplify with Dual Index adapters- TS Set A well ID. Incubate in a thermal cycler with CG000407 program based on cell cycle determined.
	Perform final SPRI cleanup; elute in Buffer EB.

QC Metric	Acceptable Range	Notes
DV200	≥30%	Tissue pre-screen
Library concentration	2–10 nM	Qubit dsDNA HS
Library size	240 bp	Bioanalyzer HS DNA chip
Reads per spot	35,000	Recommended