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Protocol: RCA on FFPE Tissue

Version: v1.0 (2026-01-29)

Reagent Safety

Use xylene and formamide in a fume hood.

1. Baking

  • 56°C for 16 hours.

2. Deparaffinization

  1. Dewaxing agent (or Xylene): 2 x 10 min.
  2. 100% Ethanol: 10 min.
  3. Methanol–acetic acid (3:1): 5 min.
  4. Graded Ethanol: 100% (5 min) -> 85% (5 min) -> 70% (5 min).
  5. Water: 3 min.

3. RNA Retrieval

  • Sodium citrate (0.01 M, pH 6): Incubate 45 min at 80°C.

4. Autofluorescence Quenching

  • 1% NaBH₄ in PBS for 30 min.
  • Note: Bubbles will form; replenish solution every 1-2 min.

5. Library Construction

(Steps similar to Fresh Frozen but with extended times)

  • Digestion: Pepsin at 37°C for 10 min.
  • Hybridization: 45°C for 120 min.
  • Ligation: 30°C for 2 hours (using SplintR).
  • RCA: 30°C for 12-16 hours.

6. Post-Amplification

  • Fix: 4% PFA.
  • Strip: 65% Formamide.
  • Bleach (Optional): Sudan Black B (0.1%) for 10 min if background is high.