Internal / Unpublished
This protocol uses iLock (invader padLock) probes, which are currently unpublished work from our lab. Please do not distribute externally.
RCA Protocol: Fresh Frozen with iLock Probes¶
- Applicable samples: Fresh Frozen
- Version: v5.4
- Last modified: 2026-01-29
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 3--5 min.
- Wash: PBS-Tween 0.05% wash 2 x 2 min.
4. Blocking¶
Goal: Block nonspecific sites.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | 58.5 μl | ||
| Ampligase Buffer | 10X | 1X | 10 μl |
| KCl | 1 M | 0.05 M | 5 μl |
| Formamide | 100% | 20% | 20 μl |
| Oligo dT | 100 μM | 1 μM | 1 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| tRNA | 10 μg/μl | 0.2 μg/μl | 2 μl |
| RiboLock | 40 U/μl | 1 U/μl | 2.5 μl |
- Incubate: Add Blocking Mix, incubate at RT for 30 min.
5. Hybridization¶
Goal: Probe-specific binding.
Probe volume
Probe stock concentration and volume depend on the specific probe panel. Adjust to achieve the desired final concentration (typically 0.05--0.1 μM per probe). Fill H₂O to 100 μl total.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | to 100 μl | ||
| Ampligase Buffer | 10X | 1X | 10 μl |
| KCl | 1 M | 0.05 M | 5 μl |
| Formamide | 100% | 20% | 20 μl |
| Probes (iLock) | (panel-dependent) | 0.05--0.1 μM | (variable) |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| tRNA | 10 μg/μl | 0.2 μg/μl | 2 μl |
| RiboLock | 40 U/μl | 1 U/μl | 2.5 μl |
- Incubate: 55°C (20 min) -> 45°C (120 min). Seal to prevent evaporation.
- Stringent Wash: 10% Formamide in 2X SSC, 3 x 10 min (45°C optimal, or RT).
- Wash: PBS-Tween 0.05% wash 2 x 1 min.
6. Activation (5' Flap Cleavage)¶
Goal: Taq DNA Polymerase cleaves the 5' non-complementary flap of iLock probes via its 5'→3' exonuclease (flap endonuclease) activity. This structure-specific cleavage activates the probe for ligation, providing an additional layer of target specificity.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | 65.5 μl | ||
| Taq DNA Pol Buffer | 10X | 1X | 10 μl |
| MgCl₂ | 50 mM | 8 mM | 16 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| Taq DNA Polymerase | 5 U/μl | 0.25 U/μl | 5 μl |
| RiboLock | 40 U/μl | 1 U/μl | 2.5 μl |
- Incubate: 45°C for 60 min (sealed).
- Wash: PBS-Tween 0.05% wash 2 x 1 min.
7. Ligation¶
Goal: Ligation of activated (flap-cleaved) iLock probes to form circles.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | 76.5 μl | ||
| SplintR Buffer | 10X | 1X | 10 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| SplintR Ligase | 25 U/μl | 2.5 U/μl | 10 μl |
| RiboLock | 40 U/μl | 1 U/μl | 2.5 μl |
- Incubate: 37°C for 2 hours (sealed).
- Wash: PBS-Tween 0.05% wash 2 x 1 min.
8. Rolling Circle Amplification (RCA)¶
Goal: Amplify signal.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | 78.5 μl | ||
| Phi29 Buffer | 10X | 1X | 10 μl |
| dNTPs | 10 mM | 0.25 mM | 2.5 μl |
| Amino-dUTP | 2 mM | 50 μM | 2.5 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| RCA Primer | 10 μM | 0.3 μM | 3 μl |
| Phi29 Polymerase | 10 U/μl | 0.25 U/μl | 2.5 μl |
- Incubate: 30°C overnight (12-16 h). (Sealed / humid chamber)
- Wash: PBS-Tween wash 2 x 1 min.
9. Post-amplification Fix¶
- Add 4% PFA, mix by gentle pipetting (3--5 times), incubate 15--30 min at RT to fix RCA products.
- Wash: PBS-Tween 0.05% wash 3 x 1 min.
- Strip: 65% formamide wash 3 x 2--10 min at 30°C (e.g. on a PCR block or Eppendorf Thermostat). Washing buffer: 650 µL formamide + 350 µL NFW.
- Wash: PBS-Tween 0.05% wash 2 x 1 min.