FFPE Protocol (2024)
Words and Solution preparation
NFW：Nuclease Free Water
****
Day 1
- Baking (~16h)
i. Attach one coverslip with a mounted FFPE section onto a microscope slide taping the edge close to the smeared slide area, where the sample name is written.
ii. Bake the tissue for 16 h at 56 °C
Day 2
NOTE: in the following steps, "Jar" denotes a step performed in a separate EasyDip™ Slide Staining Jar previously decontaminated with RNaseZap®. Unless otherwise indicated, all steps are done at room temperature
- Deparaffinization (~1h)
Jar #1: 脱蜡剂(或二甲苯), 10min * 2
Jar #2: Ethanol 100%, 10 min
Jar #3: Methanol-Acetic Acid 3:1 (v/v), 5 min
Jar #2: Ethanol 100%, 5 min
Jar #4: Ethanol 85%, 5 min
Jar #5: Ethanol 70%, 5 min
Jar #6: NFW, 3 min
- RNA retrieval (~1h)
Jar #7: Sodium citrate 0.01 M pH 6 pre-warmed at 80 °C (better + RVC 1:20)
- Incubate 45 min at 80 °C in a water bath
Jar #6: NFW, 5 min
Jar #5: Ethanol 70%, 5 min
Jar #4: Ethanol 85%, 5 min
Jar #2: Ethanol 100%, 5 min
- Air-dry tissue sections at room temperature
NOTE: at this point, tissue sections can be stored dry for several days at +4 °C
- Cover each tissue section with PDMS Chamber
****
- Rehydration (~20min)
i. Incubate in Ethanol 100% for 5 min
ii. Incubate in Ethanol 85% for 5 min
iii. Incubate in Ethanol 70% for 5 min
iv. Incubate in NFW for 5 min
- Autofluorescence quenching (~40min)
i. Incubate for 30min in NaBH₄ with strong light photobleaching.
ii. Wash the slides for 3 x 2 min with PBS-Tween 0.05%.
NaBH4: prepare 10 ml 1%(wt/vol) NaBH4 solution in 1X PBS on ice freshly each time. Exert extreme caution while weighing the powder, as accidentally dropping liquid in the stock bottle will result in an explosive reaction! Because gas bubbles form continuously in the chamber, aspirate the liquid and replenish the chamber with fresh solution every 1-2 min during the incubation
- Digestion(~20min)
i. Pepsin 0.04% in HCl 0.1 M, 10 min
ii. Wash the slides for 3 x 2 min with PBS-Tween 0.05%.
- Blocking (~30min)
| Reagents | stock | final | 1 x slide | x slides |
|---|---|---|---|---|
| NFW | ||||
| Ampligase Buffer | 10x | 1x | 10 μl | |
| KCl | 1M | 0.05M | 5 μl | |
| Formamide deionized | 100% | 20% | 20 μl | |
| Probes oligo | 10μM | 0.05μM | 1 μl | |
| BSA | 10μg/μl | 0.2μg/μl | 2 μl | |
| RiboLock (Thermo) | 40U/μl | 1U/μl | 2.5 μl | |
| tRNA (Ambion AM7119) | 10μg/μl | 0.2μg/μl | 2 μl | |
| Total | 100μl |
i. Add the blocking solution into the chamber, mix by gentle pipetting (3-5 times)
ii. incubate the slides at room temperature (RT) for 30 min.
- Hybridization of Padlock probes (~3h)
| Reagents | stock | final | 1 x slide | x slides |
|---|---|---|---|---|
| NFW | ||||
| Ampligase Buffer | 10x | 1x | 10 μl | |
| KCl | 1M | 0.05M | 5 μl | |
| Formamide deionized | 100% | 20% | 20 μl | |
| Probes | 0.2μM | |||
| BSA | 10μg/μl | 0.2μg/μl | 2 μl | |
| RiboLock (Thermo) | 40U/μl | 1U/μl | 2.5 μl | |
| tRNA (Ambion AM7119) | 10μg/μl | 0.2μg/μl | 2 μl | |
| Total | 100μl |
i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal.
ii. Incubate the slides at 55°C for 18 min for denaturation and at 45°C for 120 min for hybridization of the probes onto the target mRNA. Note: 烘箱可调62-63 °C/52-53 °C
iii. Wash the slides for 3 x 10 min with washing buffer to remove unhybridized probes.
Washing buffer: 100µL 20X SSC + 100µL formamide + 800µL NFW.
iv. Wash the slides for 3 x 1 min with PBS-Tween 0.05% to remove the remaining formamide, as it can deactivate the enzyme in the following step.
- Ligation of Padlock probes (~2h)
The ligation of the hybridized padlock probes is mediated by SplintR ligase (PBCV-1 DNA Ligase) which can function with DNA:RNA hybrid molecules.
| Reagents | stock | final | 1 x slide | x slide |
|---|---|---|---|---|
| NFW | 75.5 ul | |||
| SplintR Buffer | 10X | 1X | 10 ul | |
| BSA | 10 ug/ul | 0.2 ug/ul | 2 ul | |
| SplintR (NEB) | 25 U/ul | 2.5 U/ul | 10 ul | |
| RiboLock (Thermo) | 40 U/ul | 1 U/ul | 2.5 ul | |
| Total | 100 ul |
i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal
ii. Incubate the slides at 37 °C for 2 hours
iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.
- Rolling circle amplification (RCA, ~3h-17h)
| Reagents | stock | final | 1 x slide | x slide |
|---|---|---|---|---|
| NFW | 57 ul | |||
| Phi29 | 10uM | 1uM | 2.5 ul1 | |
| Phi29 Buffer | 10X | 1X | 10 ul | |
| Glycerol | 100% | 0.2 ug/ul | 10 ul | |
| dNTPs | 10 mM | 0.25mM | 2.5 ul | |
| NH2-dUTP | 2 mM | 50 uM | 2.5 ul | |
| BSA | 10 ug/ul | 0.2 ug/ul | 2 ul | |
| RCA Primer 1+2 | 10uM for both | 0.1uM for both | 3 + 3 ul | |
| Total | 100 ul |
i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal
ii. Incubate the slides at 30 °C for 12-16 hours (For 2h, need to incubate at 37 °C)
iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.
Day3
- Post amplification fix
i. Add 0.2% PEG9 in PBST mix by gentle pipetting (3-5 times) and incubate for 15-30 min at RT to fix the RCA products on the tissue.
Fix: 2µL PEG9 BS + 98µL PBST
ii. Wash the slides for 3 x 1 min with PBS-Tween 0.05%.
iii. Wash the slides for 3 x 2-10 min with 65% formamide at 30 °C on a PCR machine block or an Eppendorf Thermostat.
Washing buffer2: 650 µL Formamide + 350 µL NFW
iv. Wash the slides for 2 x 1 min with PBS-Tween 0.05%
- Autofluorescence bleach
Sudan B Black(storage): Sudan B Black 0.1%(wt/vol), Sudan Black B 10mg in 10ml 70% EtOH, shake in RT for days
i. Incubate for 10 min at RT with Sudan Black B.
ii. Wash the slides for 10 x 3 min with 70% EtOH.
Glycerol solution, 10% (vol/vol)
Combine 1 ml of UltraPure glycerol with 9 ml of 1× PBS in a 15-ml centrifuge tube. 10% (vol/vol) glycerol in PBS can be prepared in advance and stored at RT for several weeks.
-
This amount is 10ul for NEB phi29 and 2.5ul for Thermo phi29 ↩