跳转至

PRISM-seq组织荧光染色

（2024年12月22日更新）

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), 20min

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

注意事项：

  1. 染缸及台面清洁：Zap试剂喷洒台面------无水乙醇------纯水。

  2. 试剂封存：用毕所有试剂封口，除外脱蜡剂。大批量实验建议每次更换试剂。

  3. 提前预热烘箱，需要电子温度计。

  4. 无水乙醇需超纯。

  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

  • Cover each tissue section with PDMS Chamber

注意事项：

1. Chamber打孔时注意垂直打孔。使用时需要使用无水乙醇擦净。上表面有气泡，所以应该使用下表面贴玻片。

2. 全程组织应该保持湿润。

4. Rehydration (~20min)

i. Incubate in Ethanol 100% for 5 min

ii. Incubate in Ethanol 80% for 5 min

iii. Incubate in Ethanol 70% for 5 min

iv. Incubate in PBST for 2 min * 3次

注意事项：

1. 加在chamber中的试剂应该保存在15ml离心管中使用。

  1. Autofluorescence quenching (~40min)

i. Incubate for 6min * 5 次 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

注意事项：

1. 硼氢化钠加的间隙需放置在4度，加在组织上会有气泡冒出。

2. PBS需要提前放在4度。

  1. Digestion(~20min)

i. Pepsin 0.04% in HCl 0.1 M, 5 min 在37度。

ii. Wash the slides for 3 x 2 min with PBS-Tween 0.05%.

注意事项：

1. 下一步试剂提前拿出来化冻。

  1. Blocking (~30min)
Reagents stock final 1 x slide x slides
NFW 57.5ul
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 chambe

ii. incubate the slides at room temperature (RT) for 30 min.

注意事项：

1. 所有试剂加之前需要vortex（除了酶），加至只剩酶时vortex；酶不能握住管子，不能vortex, 应轻轻吹匀，混合后也应轻轻混匀。

2. 预热烘箱。

8. Hybridization of Padlock probes (~3h)

Reagents stock final 1 x slide x slides
NFW 32.1ul
Ampligase Buffer 10x 1x 10 μl
KCl 1M 0.05M 5 μl
Formamide deionized 100% 20% 20 μl
Probes 0.2μM 26.4ul
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 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.

注意事项：

1. 试剂混合方法注意点和上一步同。注意酶的使用。

2. Seal时注意用无纺布沾湿无水乙醇擦干chamber，seal时注意留一条缝。

3. 甲酰胺避光保存。

9. 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, 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%.

注意事项：

1. SplintR和Ribo是两个酶，注意使用它。

2. SplintR buffer室温下容易出现絮状物，注意振荡和用体温溶解。

3. Seal时注意每一步都需要用无水乙醇搽干净Chamber.

10. Rolling circle amplification (RCA, ~3h-17h)

Reagents stock final 1 x slide x slide
NFW 67.5 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 Mix 10uM for both 0.1uM for both 3 ul
Total 100 ul

i. Add the solution into the chamber 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%.

注意事项：

1. 这一步12个小时最好。

2. 甘油吸取和打液时都要慢，注意足量，否则荧光过强。

3. 试剂再加入Phi29(酶)之前需要混匀，因为有甘油所以不能震荡，只能用枪慢慢吹匀。加入Phi29之后也只能用枪慢慢混匀。

4. 过夜孵育后洗涤，注意一定要慢，吸液和加液都要慢，否则会把扩增球体吹散。

Day3

11. Post amplification fix

i. Add 4% PFA and incubate for 30 min at RT to fix the RCA products on the tissue.

ii. Wash the slides for 3 x 1 min with PBS-Tween 0.05%.

iii. Wash the slides for 3 x 10 min with 65% formamide at RT.

Washing buffer2: 650 µL Formamide + 350 µL NFW

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

注意事项：

1. 可以加入PBST之后放入4度冰箱暂存。

12. 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 8-10 x 3 min with 70% EtOH.（直到液体变成清澈透明）

iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%（观察到组织变黑）

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.

注意事项：

1. 苏丹黑，不会完全溶解，取用时吸取中层，不要震荡。

13. 荧光染色（~1.5h）

i. 全程避光操作。

ii. 烘箱提前开启。

iii. 每个组织加入荧光探针100ul。

iv. 50度5min；再37度15min或更长。（注意放在盛有液体的枪头盒上，枪头盒上裹上锡纸，保持组织湿润）

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

vi. DAPI每片加入100ul，RT反应5min。枪头盒盖上锡纸。

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

viii. 去掉Chamber，擦干组织周围，加入25ul 10%的甘油，盖上干净的盖玻片，注意组织周围不要有气泡。

ix. 封片：四周刷一层指甲油，等20min干，注意全程放在4度玻片盒。

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