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H&E Staining

Morphology staining performed after signal readout (PRISM imaging or SPRINTseq sequencing) and fluorescent morphology staining (DAPI/WGA). Provides standard histological context in brightfield.

In Flow Cell (SPRINTseq)

Goal: Complete H&E staining inside the Flow Cell. Do not use xylene.

Warning

All reagents must be pre-filtered (0.2--0.45 μm) to avoid clogging the flow path.

  1. Wash: Pump ample PBS to remove residual sequencing reagents (glycerol/fluorophores). Flow rate: 100--200 μl/min.
  2. Hematoxylin: Pump Mayer's Hematoxylin, stop flow and incubate 2--5 min.
    • Observe Flow Cell color change.
  3. Rinse: Pump DI Water or weakly acidic water until effluent runs clear.

  4. 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.

  5. Bluing: Pump Scott's Tap Water Substitute (or PBS), stop flow and incubate 1 min.

  6. Eosin: Pump Eosin Y, stop flow and incubate 30 s -- 1 min.
  7. 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).
  8. Imaging medium: Pump PBS or index-matching fluid (water-soluble) to fill Flow Cell.
    • Do not air-dry. Image in brightfield under liquid.

On Slide (PRISM / Manual)

For slides not in a flow cell, standard histology H&E can be performed:

  1. Wash: PBS, 2 x 2 min.
  2. Hematoxylin: Immerse in Mayer's Hematoxylin, 5--8 min.
  3. Rinse: Running tap water, 5 min.
  4. Differentiation: 0.5% HCl in 70% EtOH, quick dip (2--5 s).
  5. Bluing: Scott's Tap Water or running tap water, 1--2 min.
  6. Eosin: Eosin Y, 30 s -- 2 min.
  7. Dehydrate: 95% EtOH → 100% EtOH → xylene (or xylene substitute).
  8. Mount: DPX or similar permanent mounting medium.
  9. Image: Brightfield microscopy.

Reagents

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