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1Repair Windows errors before they cause bigger problems2Fix the driver behind crashes, sound loss and screen glitches3Clear out junk files and repair common Windows errorsUse the preservation method validated for your exact spatial assay and the specimen you actually have. Fresh-frozen tissue suits workflows designed for it and requires careful freezing and section handling. FFPE can make archived clinical material usable, but RNA quality depends on fixation and storage and compatibility is chemistry-specific. Fixed-frozen tissue is a separate, protocol-specific category—not a synonym for either fresh-frozen or FFPE.
Start with the specimen and assay you can actually use
If you have an archived FFPE block
Choose a workflow whose current documentation explicitly supports FFPE. A fresh-frozen-only chemistry cannot be assumed to work on a paraffin block. FFPE can provide access to historical or biobanked clinical specimens, but RNA integrity may vary with preparation and archival history. See 10x Genomics’ FFPE tissue preparation guide and Visium FFPE tissue-preparation documentation.
If you are collecting new tissue
Choose the assay and preservation plan together, before harvesting. The appropriate method depends on the assay’s validated chemistry, the species and tissue, and the biological readout you need—not just on whether a platform name appears on the box.
If the specimen is fixed-frozen
Check for a protocol specifically validated for that preparation. One 10x CytAssist workflow describes mouse tissue fixed in PFA, sucrose-cryopreserved, embedded in OCT, and processed with probe-based chemistry. That example does not establish compatibility for other tissues or assays; consult the fixed-frozen workflow documentation for its stated scope.
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What the preservation choice changes
| Decision point | Fresh-frozen | Fixed tissue, including FFPE | What to check |
|---|---|---|---|
| Typical specimen situation | Newly collected tissue or a suitably frozen block | Often an archived clinical or biobanked specimen | Which material is available and whether the assay supports it |
| RNA and morphology | Prompt, controlled freezing helps protect RNA; ice-crystal formation and handling can affect morphology | Fixation can preserve morphology, while RNA integrity may be compromised and can vary with storage history | Assess the actual specimen rather than relying on its preservation label |
| Assay chemistry | Some workflows capture polyadenylated RNA and specify fresh-frozen tissue | Specified probe-based workflows can support FFPE or certain fixed-frozen preparations | Exact product version, chemistry, tissue, species, and protocol |
| Preparation demands | Collection, freezing, embedding, cryosectioning, and assay-specific optimization | Fixation and embedding or fixed-frozen preparation; section adhesion and RNA quality matter | Current tissue-preparation and quality-control guidance |
| Potential readout | Some sequencing-based workflows offer broad transcriptome capture | Imaging assays profile designed probe panels in situ; probe-based sequencing workflows also exist | Panel breadth, spatial readout, imaging needs, and analysis requirements |
This is a qualitative comparison, not evidence that one preservation method universally produces more genes, finer resolution, or better biological conclusions.
Fresh-frozen: suitable when handling is controlled
Fresh-frozen tissue avoids formalin crosslinking and is appropriate for workflows validated for fresh-frozen material. It is not automatically high quality: delays after collection, freezing and embedding choices, storage, and sectioning can all affect RNA and tissue morphology. For Visium HD, the 10x Genomics Visium HD Fresh Frozen Tissue Preparation Handbook 2.0, updated July 31, 2026, describes prompt snap-freezing or direct embedding in OCT to limit RNA degradation and ice-crystal formation. OCT provides support during cryosectioning; unvalidated alternatives should not be presumed equivalent.
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That handbook calls an RNA integrity number (RIN) of 4 or higher optimal for Visium HD, but explicitly notes that a lower RIN does not necessarily result in poor data. Treat the figure as assay-specific workflow guidance, not a universal pass/fail cutoff. The Visium HD tested-tissues guidance, updated July 20, 2026, also notes that biology, freezing, embedding, and block handling affect performance and recommends assessing tissue RNA quality.
- Minimize post-mortem delay and use RNase-free handling.
- Freeze promptly and control sectioning conditions.
- Follow the exact assay’s quality checks and optimize permeabilization when required by the workflow.
- Use tissue-specific preparation guidance where available; the Visium tissue-preparation guide describes workflow considerations for fresh-frozen tissue.
Fixed tissue: distinguish FFPE from fixed-frozen
FFPE
Formalin-fixed, paraffin-embedded tissue is widely available from pathology archives and can preserve recognizable tissue structure. The trade-off is that RNA integrity can be impaired and may decline with extended archival storage. A protocol designed for FFPE may use probes to detect defined sequences rather than relying on the same capture strategy as a fresh-frozen poly-A workflow. That does not remove the need to check assay-specific quality requirements, section adhesion, and the condition of the specimen.
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Fixed-frozen
Fixed-frozen tissue has its own preparation and validation requirements. The cited 10x CytAssist example uses PFA fixation, sucrose cryoprotection, OCT embedding, and probe-based chemistry for mouse tissue. Do not infer from that example that any fixed-frozen sample is compatible with any Visium, CytAssist, or Xenium workflow.
Platform labels alone are not enough to determine compatibility. 10x Genomics’ spatial platform support overview distinguishes workflow types; check the current protocol and product version, species, tissue, and kit before committing a specimen.
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Choose the readout that fits the biological question
Preservation is only one part of the decision. Sequencing-based capture can provide broader transcriptome profiling in workflows that support it. Imaging-based assays use designed probe panels and image transcripts in place, which can support cellular localization but constrains measurement to the panel and brings platform-specific imaging and analysis trade-offs. Consider these factors before choosing the preservation workflow:
- Whether you need broad transcriptome discovery or a targeted set of genes.
- How much cellular localization the question requires.
- Whether the assay’s panel design includes the relevant targets.
- The imaging, segmentation, and downstream analysis demands of the selected platform.
The 2025 Nature Communications imaging-platform benchmark compared Xenium, Vizgen MERSCOPE, and NanoString CosMx on FFPE tissue microarrays covering 17 tumor and 16 normal tissue types. In that study, Xenium generated higher transcript counts per matched gene; the platforms also differed in clustering behavior, false discovery rates, and cell-segmentation errors. These findings concern three imaging platforms on FFPE samples, not a controlled comparison of fresh-frozen against fixed tissue, and they do not establish a general preservation winner. A separate peer-reviewed discussion of spatial methods likewise describes differing measurement strategies rather than establishing one preservation method as best across platforms and tissues: Goods et al., peer-reviewed article.
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A practical selection sequence
- Inventory the specimen. Establish whether you have an archived FFPE block, fresh tissue to collect, a frozen block, or fixed-frozen material.
- Identify the exact assay and version. Confirm that its current documentation accepts that preservation type, species, and tissue; do not decide from “Visium” or “spatial transcriptomics” alone.
- Check specimen quality and preparation requirements. Review RNA quality guidance, section adhesion and handling requirements, and any tissue-specific recommendations. A preservation category by itself does not guarantee success.
- Match the readout to the experiment. Decide whether broad capture or a defined imaging panel best answers the biological question, and account for resolution and analysis trade-offs.
- Plan handling around the selected workflow. For fresh-frozen material, coordinate collection, freezing, embedding, and sectioning. For fixed workflows, follow the specified fixation, embedding, and QC steps. Where feasible, assess the actual tissue before processing the full study.
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