Skip to content

Protocol: RCA on Fresh Frozen Tissue

Version: v5.3 (2026-01-29)

Applicability

Samples: Mouse Brain and most fresh frozen tissues.

1. Sample Preparation & Sectioning

Goal: Obtain high-quality sections free of ice crystals and cracks.

  1. Embedding: Use isopentane + dry ice for rapid freezing.
  2. Cryosectioning:
    • Blade: -20°C
    • Head: -10°C
    • Thickness: 10 µm
  3. Section Placement: Pick up on pre-chilled slide -> finger-back press -> return to cold stage.
  4. Storage: -80°C.

2. Fixation

  1. Thaw: Place slide immediately on 37°C hot plate for 1 min.
  2. Fix: Immerse in 4% PFA (Fresh) for 30 min at RT.
  3. Wash: PBS wash 2 x 2 min.

3. Permeabilization

  1. Incubate: 0.04% Pepsin (in 0.1 M HCl) at 37°C for 2 min.
  2. Wash: PBS-Tween 0.05% wash 2 x 2 min.

4. Dehydration

Goal: Improve tissue permeability.

  1. 80% Ethanol: 10 min.
  2. 100% Ethanol: 2 min.
  3. Rehydrate: PBS-Tween 0.05% wash 2 x 2 min.

5. Blocking

Blocking Mix (100 µl): * Ampligase Buffer (1X) * KCl (0.05 M) * Formamide (20%) * Oligo dT (1 µM) * BSA (0.2 mg/ml) & tRNA (0.2 mg/ml) * RiboLock (1 U/µl)

Action: Incubate at RT for 30 min.

6. Hybridization

Conditions: * 55°C for 15 min -> 45°C for 120 min. * Seal chamber to prevent evaporation.

Wash: * Stringent Wash: 10% Formamide in 2X SSC, 3 x 10 min at 45°C. * PBS-T wash 2 x 1 min.

7. Ligation

  • Enzyme: SplintR Ligase (2.5 U/µl).
  • Incubate: 37°C for 2 hours (sealed).

8. Rolling Circle Amplification (RCA)

  • Enzyme: Phi29 Polymerase (0.25 U/µl).
  • Incubate: 30°C Overnight (12-16 h).

9. Post-Amplification Fix

  1. Fix: 4% PFA for 15-30 min at RT.
  2. Strip: 65% Formamide wash at 30°C (3 x 2-10 min).