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Sample Pre-treatment: Fresh Frozen

  • Applicable samples: Fresh Frozen (FF) cryosections
  • Version: v5.3
  • Last modified: 2026-01-29

What's next?

After completing pre-treatment, proceed to the unified RCA Protocol (Blocking → Hybridization → Ligation → RCA → Post-fix).

1. Sample Preparation & Sectioning

Goal: Obtain high-quality sections free of ice crystals and cracks, with firm adhesion to slides.

  • Embedding & Freezing: Use isopentane + dry ice for rapid freezing.
  • Cryosectioning: Blade: -20°C, Head: -10°C, Thickness: 10 µm.
  • Section Placement: Pre-chilled slide pickup -> finger-back press for adhesion -> return to cold stage immediately.
  • Storage: Store at -80°C.

2. Fixation

Goal: Brief thermal retrieval for adhesion, then chemical fixation.

  • Retrieve: Transport slides on dry ice.
  • Thaw & Adhere: Place immediately on 37°C hot plate for 1 min.
  • Fixation: Immerse vertically in Slide Mailer with 4% PFA (Fresh), incubate at room temperature (RT) for 30 min.
  • Wash: PBS wash 2 x 2 min.

Assemble Chamber: Assemble pre-treatment chamber to limit liquid mount.

3. Permeabilization

Goal: Mild pore formation.

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

4. Dehydration

Goal: Improve tissue permeability.

  • Pre-dehydrate: 80% Ethanol for 10 min.
  • Dehydrate: 100% Ethanol for 2 min.
  • Rehydrate: PBS-Tween 0.05% wash 2 x 2 min.

Next step: RCA Protocol (Blocking → RCA)