H&E Staining¶
- Applicable samples: (A) clean FFPE sections, e.g. the section adjacent to a spatial run; (B) the same section after signal readout (PRISM imaging or SPRINTseq sequencing), FFPE or fresh frozen; (C) a section still in the SPRINTseq flow cell
- Version: v2.0
- Last modified: 2026-09-26
Brightfield histology for the morphological context of a spatial run. The staining itself does not depend on what the section has been through; how the section gets to water does. Pick the entry route by where the section starts, then run the shared staining core (Sections 4–6).
| Where the section starts | Entry route | Then |
|---|---|---|
| Clean FFPE section, still in paraffin | 2. Route A — bake, dewax, rehydrate | Sections 4–7 |
| Same section after PRISM / SPRINTseq readout — already dewaxed, hydrated | 3. Route B — no bake, no dewax | Sections 4–7 |
| Section still assembled in the SPRINTseq flow cell | 9. Variant C | Self-contained |
After readout, H&E is the last thing that happens to the section
Eosin is strongly fluorescent and hematoxylin absorbs across the visible range, so no fluorescence imaging is possible afterwards. Finish and check every fluorescence acquisition first — including the DAPI scan that the H&E image will be registered to.
Adapted from
The staining core (Sections 4–6) is the lab's own FFPE H&E bench protocol. Route B follows 10x Genomics' post-Xenium H&E demonstrated protocol1 and post-Xenium technical note2, adapted to PRISM/SPRINTseq sections: the section enters the stain hydrated, with no bake and no dewax. 10x's Quencher Removal step is deliberately not carried over (Section 3).
1. Before You Start¶
Goal: Jars filled and labelled, so the staining core runs without pauses.
- Jars (one per solution; the core reuses none): dewaxing agent (route A only), 100% ethanol × 4, 95% ethanol × 4, 70% ethanol × 2, Milli-Q water × 4, hematoxylin, bluing reagent, eosin, clearing agent × 2.
- Ethanol grades made fresh from absolute ethanol and Milli-Q water.
- Mounting medium and coverslips at hand before the clearing steps — the section must not dry between the last clearing jar and the coverslip.
- Route B only: the section's fluorescence data has been checked (warning above), and PBS is ready for the coverslip soak.
Safety
Dewaxing and clearing agents are flammable solvents: work in a fume hood, away from heat sources, and collect the waste as organic solvent. Hematoxylin and eosin stain skin and bench surfaces — gloves and a lab coat.
2. Route A: Clean FFPE Section (~70 min)¶
Goal: Melt and remove the paraffin, then bring the section down to water.
- Bake: 60 °C, 30 min.
- Dewax: plant-based dewaxing agent, 30 min.
- Rehydrate:
- 100% ethanol, 2 min — jar 1.
- 100% ethanol, 2 min — jar 2.
- 95% ethanol, 2 min.
- 70% ethanol, 2 min.
- Continue at 4. Hematoxylin and Bluing.
This bake is not the pre-treatment bake
60 °C for 30 min only softens the paraffin ahead of dewaxing. The 56 °C, 16 h bake in Pre-treatment: FFPE §1 is for adhesion through days of on-slide chemistry. H&E on a clean section does not need that.
3. Route B: Same Section After Readout (~15 min)¶
Goal: Get an already-dewaxed, hydrated section into water without letting it dry.
Do not bake. Do not dewax.
The paraffin came off in pre-treatment, and the section has been hydrated ever since. The dewax series has nothing to remove, and baking would dry the section — drying before H&E is what 10x blames for tissue shrinkage1. Route B joins the staining core at its first water step.
Stain within 3 days, and keep the section wet until then
10x validates post-run H&E only within 3 days of the end of the run, with the slide held in PBS-T (0.05%) at 4 °C12, and attributes tissue shrinkage to the section drying between the run and the stain1. How long a PRISM section keeps under its sealed 10% glycerol mount at 4 °C has not been measured (TODO) — treat 3 days as the target.
- Check first: all fluorescence data acquired and checked, including the DAPI scan (warning at the top of the page).
- PRISM — cut the seal: run a razor blade through the nail-polish ring along the coverslip edge, without pressing on the coverslip.
- PRISM — float the coverslip off: immerse the slide in PBS until the imaging coverslip slides free, then lift it away. Never pry — the tissue sits between the section coverslip and the imaging coverslip, and prying tears it.
- Wash out the glycerol: dip in Milli-Q water about 30 times (10x's decoverslipping wash2).
- SPRINTseq, off the flow cell: disassemble the flow cell with the section kept wet — procedure TODO. To stain inside the flow cell instead, use Variant C.
- Continue at 4. Hematoxylin and Bluing, from its first water step, using the route B hematoxylin time.
No Quencher Removal step
10x's post-run protocol opens with 10 mM sodium hydrosulfite for 10 min, then 3 × 1 min water1. That step strips 10x's own proprietary autofluorescence reagent, which our workflow does not use — do not add it by analogy. Our quenchers behave differently: NaBH₄ and photobleaching leave nothing visible, while Sudan Black B and TrueBlack (both applied in 70% ethanol) are partly extracted by the ethanol steps here and can leave grey-black granules (Section 8).
The section is on a coverslip taped to a carrier slide
See Pre-treatment: FFPE §1. Whether the tape adhesive survives the ethanol series and the terpene clearing agent, and whether to finish by mounting the section coverslip tissue-down on a fresh slide, is TODO — settle it on the first route B section.
Planning H&E already at imaging time
The nail-polish seal is what makes decoverslipping risky. 10x uses an unsealed, water-soluble mount on sections that will be decoverslipped and stained later2, and its Visium H&E protocol rules out nail polish wherever the coverslip must come off again3. Whether a PRISM imaging session tolerates an unsealed coverslip is TODO.
4. Hematoxylin and Bluing (~20 min)¶
Goal: Stain nuclear chromatin, then turn it from red-purple to blue.
- Water: Milli-Q water, 2 min.
- Hematoxylin: 10 min (route A) — the starting time; extend it if nuclei come out pale (note below). Route B: start at 20 min, the time 10x validates on post-run sections1. Record the time used.
- Rinse: Milli-Q water, 3 × 1 min (three jars).
- Bluing: bluing reagent, 1 min.
- Rinse: Milli-Q water, 1 min, 2 min, 2 min (three jars).
Progressive staining, no differentiation — because our nuclei stain weakly
Nuclei in our sections tend to take up hematoxylin weakly; the cause is not established (pH has been suspected). That decides the method:
- Regressive staining overstains, then strips the excess with acid alcohol (0.5% HCl in 70% ethanol). On weak nuclei the differentiation takes away stain we cannot spare.
- Progressive staining builds colour up and stops at nuclear chromatin by itself, so nuclei are deepened simply by leaving the section in hematoxylin longer.
So there is no acid-alcohol step, and the lever for pale nuclei is time, not a different reagent. Extend the hematoxylin and judge at the microscope: stop when nuclei are dark with chromatin still visible, before the cytoplasm and background pick up a blue cast. Do not swap in a regressive (Harris-type) hematoxylin here — without differentiation it overstains everything.
After readout, expect to need longer still. 10x also stains post-run sections progressively, with no differentiation, and holds the same Mayer's hematoxylin (MHS16) 20 min1 — against 3 min on an unprocessed FFPE section in its Visium protocol3. The delivery differs (immersion vs. 1 ml on the slide), so read that as a direction, not a ratio. 10x also notes hematoxylin pH drifts with age: use fresh solution1.
5. Eosin (~5 min)¶
Goal: Counterstain cytoplasm and extracellular matrix in graded shades of pink.
- Up-grade: 70% ethanol, 1 min → 95% ethanol, 1 min.
- Eosin: 0.5% alcoholic eosin Y, 1 min.
- Differentiate: 95% ethanol, 30 s — jar 1, then 30 s — jar 2.
Eosin intensity is set in the 95% rinses
The eosin is alcohol-based, which is why the section goes up to 95% ethanol before it rather than straight in from water. The two 95% rinses after it pull excess eosin: more time there gives paler, better-separated pinks.
6. Dehydrate, Clear and Mount (~10 min)¶
Goal: Remove all water, clear the tissue, and coverslip before anything dries.
- Dehydrate: 100% ethanol, 1 min — jar 1, then 1 min — jar 2.
- Clear: plant-based clearing agent, 3–5 min — jar 1, then 3–5 min — jar 2.
- Mount: mounting medium and coverslip immediately from the second clearing jar. Avoid bubbles over the tissue.
A milky clearing jar means water got through
If the clearing agent turns cloudy, water was carried over. Go back to fresh 100% ethanol, then fresh clearing agent. Mounting over residual water leaves a haze that does not come out.
7. Imaging and Registration¶
Goal: A brightfield image that can be placed on the spatial data.
- Let the mount set before scanning (10x: 30 min at RT, then image within 3 days, 4 °C meanwhile1).
- Brightfield whole-slide scan: 20X for pathological assessment, 40X or higher if the H&E image will be used for cell segmentation2.
- Route B — registration: the H&E image registers to the run through its nuclei, hematoxylin-stained nuclei against the DAPI image acquired before H&E. Fit an affine transform from at least 3 matched landmarks, then check it by overlaying DAPI nuclei on the hematoxylin nuclei across the whole section, not only near the landmarks.
8. Expected Results and QC¶
Goal: Know what a good slide looks like before judging a bad one.
| Look | Meaning | Fix next time |
|---|---|---|
| Nuclei crisp blue-purple, chromatin detail visible | Good | — |
| Nuclei pale | Common on our sections (Section 4 note); or hematoxylin exhausted | Longer hematoxylin; fresh solution if it has been reused. Do not add a differentiation step |
| Nuclei red-purple rather than blue | Bluing incomplete | Fresh bluing reagent; do not shorten the rinses after it |
| Eosin flat, one shade of pink | Over-eosinated or under-differentiated | Longer 95% rinses after eosin |
| Hazy or milky under the coverslip | Water carried into clearing | See Section 6 warning |
| Grey-black granules (route B) | Residual Sudan Black B / TrueBlack from AF quenching | Note it; not tissue pigment |
On a route B section, expect staining intensity to differ from a clean section of the same block: heat retrieval and protease digestion change how chromatin and cytoplasm take up dye. Judge the times on the first slide rather than assuming they carry over.
9. Variant C: In the SPRINTseq Flow Cell¶
Goal: Complete H&E staining inside the flow cell. Do not use xylene.
Carried over unchanged from v1.0
This variant predates the on-slide routes above, uses different reagents (Mayer's hematoxylin with acid-alcohol differentiation, aqueous eosin), and has not been checked against them.
Warning
All reagents must be pre-filtered (0.2--0.45 μm) to avoid clogging the flow path.
- Wash: Pump ample PBS to remove residual sequencing reagents (glycerol/fluorophores). Flow rate: 100--200 μl/min.
- Hematoxylin: Pump Mayer's Hematoxylin, stop flow and incubate 2--5 min.
- Observe Flow Cell color change.
- Rinse: Pump DI Water or weakly acidic water until effluent runs clear.
-
Acid Alcohol (differentiation): Quickly pump 0.5% HCl in 70% Ethanol, contact tissue 2--5 s, then immediately pump ample PBS to stop reaction.
Critical timing
Do not stop flow too long during acid alcohol differentiation. Over-differentiation removes nuclear stain.
-
Bluing: Pump Scott's Tap Water Substitute (or PBS), stop flow and incubate 1 min.
- Eosin: Pump Eosin Y, stop flow and incubate 30 s -- 1 min.
- Wash/Clear: Pump 95% Ethanol to wash.
- Note: Do not use xylene in the Flow Cell. Use multiple high-concentration ethanol washes instead (may be less perfect than xylene, but required to protect the Flow Cell).
- Imaging medium: Pump PBS or index-matching fluid (water-soluble) to fill Flow Cell.
- Do not air-dry. Image in brightfield under liquid.
Reagents¶
Routes A and B (Sections 2–6):
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| Plant-based dewaxing agent | TODO | TODO | Xylene substitute. Route A only |
| Hematoxylin (progressive) | TODO | TODO | 10 min, no differentiation (Section 4 note) |
| Bluing reagent | Biotopped | TODO | |
| Eosin Y, 0.5% alcoholic | TODO | TODO | |
| Plant-based clearing agent | TODO | TODO | Xylene substitute |
| Mounting medium | TODO | TODO | Must be compatible with the clearing agent |
Variant C (Section 9):
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| Mayer's Hematoxylin | Sigma-Aldrich | MHS16 | Pre-filter before flow cell use |
| Eosin Y (aqueous) | Sigma-Aldrich | HT110116 | Pre-filter before flow cell use |
| Scott's Tap Water Substitute | Sigma-Aldrich | S5134 | Bluing reagent |
| Acid Alcohol | -- | -- | 0.5% HCl in 70% EtOH; prepare fresh |
Ordering: Bill of Materials → H&E.
See also: Nuclear Staining (DAPI) | Cell Membrane Staining (WGA)
Changelog¶
- v2.0 -- 2026-08-15 -- Rebuilt around two entry routes and a shared staining core taken from the lab's FFPE H&E bench protocol. MAJOR: the on-slide hematoxylin changed from Mayer's 5–8 min with acid-alcohol differentiation to a progressive 10 min stain with no differentiation, a commercial bluing reagent and 0.5% alcoholic eosin; xylene is replaced by plant-based dewaxing and clearing agents. Slides stained under v1.0 and v2.0 are not directly comparable.
Why no differentiation. Nuclei in the lab's sections tend to stain weakly (cause not established; pH suspected). Acid-alcohol differentiation removes nuclear stain that these sections cannot spare, whereas a progressive stain can be deepened by extending the hematoxylin time. 10 min is the starting point, not a ceiling.
Why two routes. v1.0 covered only sections that had already been through readout, and its on-slide route had no bench record behind it. The lab's first recorded H&E was on a clean FFPE section adjacent to a spatial run (bench date 2026-08-15); that protocol is now the staining core (Sections 4–6). Same-section H&E after readout is expected to become routine, and such a section differs only in how it reaches water — it is already dewaxed and must never dry — so it enters the same core by route B, which skips the bake and the dewax. Route B follows 10x Genomics' post-Xenium H&E guidance (CG000613 Rev B, CG000709 Rev D): stain within 3 days, keep the section wet, start hematoxylin at 20 min. 10x's sodium-hydrosulfite Quencher Removal is not carried over — it targets a reagent we do not use. Route B has not yet been run in-house.
Unchanged. The flow-cell variant (now Variant C) is carried over verbatim.
- v1.0 -- 2026-08-18 -- First versioned release of this page. The
staining procedure is unchanged (page content dates to 2026-04-04); the metadata block
and this changelog were added so the page conforms to the house format and appears in
the protocol index. See docs/CONTRIBUTING.md for the format.
References¶
- 10x Genomics. Xenium In Situ Gene Expression – Post-Xenium Analyzer H&E Staining. Demonstrated Protocol CG000613, Rev B (December 2023). PDF. — route B: time window, keeping the section wet, the Quencher Removal step we omit, post-run hematoxylin time, mounting.
- 10x Genomics. Post-Xenium In Situ Applications: Immunofluorescence, H&E, Visium v2, and Visium HD. Technical Note CG000709, Rev D (August 2025). PDF. — post-run storage, decoverslipping, imaging magnification.
- 10x Genomics. Visium CytAssist Spatial Gene Expression for FFPE – Deparaffinization, H&E Staining, Imaging & Decrosslinking. Demonstrated Protocol CG000520, Rev C (April 2024). PDF. — H&E on an unprocessed FFPE section, for comparison.