FFPE (CUBIC-L)

Words and Solution preparation

Basic:

NFW：Nuclease Free Water

PBST: 1xPBS with 0.05% Tween20

Pretreatment:

Xylene

Ethanol 100%

Ethanol 85%

Ethanol 70%

Mili-Q water

Sodium Citrate: Sodium citrate 0.01 M pH 6 pre-warmed at 80 °C (better + RVC 1:20)

Photo Bleaching

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Day 1

  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

  1. 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

  1. 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

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  1. 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

  1. Autofluorescence quenching (~90min)

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

  1. 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%.

  1. 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.

  1. 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 55oC for 18 min for denaturation and at 45oC for 120 min for hybridization of the probes onto the target mRNA. Note: 烘箱可调62-63 oC/52-53 oC

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.

  1. 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 oC for 2 hours

iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.

  1. 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 oC for 12-16 hours (For 2h, need to incubate at 37 oC)

iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.

Day3

  1. 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 oC 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%

  1. Autofluorescence bleach

Lipid removal

i. Incubate for 10 min at RT with Cubic L.

ii. Wash the slides for 3 x 10 min with PBST.

Sudan B Black 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.

  1. This amount is 10ul for NEB phi29 and 2.5ul for Thermo phi29 ↩