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In-situ Sequencing & Post-Sequencing Staining

1. Sequencing / Imaging

  • Flow Cell Assembly: Assemble slide into Flow Cell at this stage (if not done earlier).
  • In-situ Sequencing Cycles: Complete all sequencing cycles (Cycle 1-10).

2. Morphology Staining

Goal: After sequencing and before H&E, pump DAPI and WGA (optional) via pump system while avoiding line contamination.

Reagent mix (pre-mix):

  • PBS: 10 mL (adjust per line volume)
  • DAPI: 5 μg/mL
  • WGA-Alexa 488: 5 μg/mL (or 10 μg/mL)

Automated workflow:

  • Wash (pre-clean): Pump PBS-T (0.05% Tween), flush 2 min to remove residual sequencing reagents.
  • Stain: Pump DAPI/WGA Mix until Flow Cell is filled.
  • Action: Stop flow.
  • Incubate: RT for 15 min.
  • Wash (post-clean): Pump PBS, flush 2 min.
  • Image: Full slide scan in DAPI and FITC (or Cy5) channels.
  • Line cleaning (critical): After imaging to prevent WGA adhering to lines.
  • Pump 4X SSC (or 0.5 M NaCl) high-salt solution, flush 1 min.
  • Pump PBS-T (0.1% Tween), flush 1 min.
  • This step keeps lines clean for subsequent H&E or next run.

3. Post-Sequencing H&E

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

Warning

All reagents must be pre-filtered to avoid clogging the flow path.

  • Wash: Pump ample PBS (or sequencer Wash Buffer) to remove sequencing reagents (glycerol/fluorophores). Recommended 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, critical): Quickly pump 0.5% HCl in 70% Ethanol, contact tissue 2-5 s, then immediately pump ample PBS to stop reaction.
  • Flow Cell challenge: timing is critical; do not stop flow too long.
  • 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 here. Use multiple high-concentration ethanol washes to remove SBB as much as possible (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.