欢迎您, 登录 | 注册

首页 | English

X
加载中

In situ hybridization is routinely used to examine the gene expression level and location of embryos. This protocol is modified from the Thisse protocol and is a detailed description of the in situ hybridization procedures in zebrafish embryos.

 

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] In Situ Hybridization (From the Thisse Method)
[Bio101] 原位杂交(源自Thisse方法)

细胞生物学 > 细胞染色 > 核酸
作者: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
2/5/2012, 6184 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.179

[Abstract] In situ hybridization is routinely used to examine the gene expression level and location of embryos. This protocol is modified from the Thisse protocol and is a detailed description of the in situ hybridization procedures in zebrafish embryos.

 

[Abstract] 原位杂交是经常用于研究基因表达水平及胚胎的位置。该方案是Thisse改良方案,详细描述斑马鱼胚胎原位杂交程序。

Materials and Reagents

  1. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
  2. PFA (powder) (Thermo Fisher Scientific)
  3. DEPC (Sigma-Aldrich, catalog number: D5758 )
  4. Glutaradehyde (Sigma-Aldrich, catalog number: G5882 )
  5. Roche anti-DIG AP (Roche Diagnostics, catalog number: 11093274910 )
  6. Torula yeast RNA (Sigma-Aldrich, catalog number: R6225 )
  7. Heparin (Sigma-Aldrich, catalog number: H0777 )
  8. Lamb/Sheep Serum (Thermo Fisher Scientific, catalog number: 16070-096 )
  9. Formamide (High purity grade)
  10. Methanol
  11. Sodium Citrate
  12. EDTA
  13. NaCl
  14. KCl
  15. MgCl2
  16. Na2HPO4
  17. KH2PO4 
  18. HCl
  19. Tween 20
  20. Citric acid
  21. 1x PBT(made from DEPC H2O) (see Recipes)
  22. Pronase (Roche Diagnostics, catalog number: 10165921001 ) (see Recipes)
  23. NBT/BCIP color substrate (Promega Corporation, catalog number: S3771 ) (see Recipes)
  24. Hybe buffer (see Recipes)
  25. Hybe- buffer (see Recipes)
  26. 20x SSC (see Recipes)
  27. 10x PBS (see Recipes)
  28. 4% paraformaldehyde/PBS (see Recipes)
  29. Blocking solution (see Recipes)
  30. Stop solution (see Recipes)
  31. Heat inactivated lamb serum (see Recipes)
  32. Pre-staining buffer (see Recipes)

Equipment

  1. Hybridization Incubator
  2. 1.5 ml tube
  3. 24-well plate
  4. Aluminum foil
  5. Nalgene filter

Procedure

  1. Preparation of embryos
    1. Dechorinate the embryos using pronase. (Add 1 drop of pronase solution to 1 ml of E3). Keep the embryos at room temperature (RT) for 3-5 min (do not let the embryos in pronase solution for too long!).
    2. Pipet the embryos to break the chorion. 
    3. Collect the embryos in 1.5 ml tubes (usually 20 embryos/per tube). 
    4. Remove E3 buffer and rinse embryos with 1 ml of new E3 buffer to get rid of pronase.
    5. Remove all the E3 and add 500 μl of 4% PFA to fix the embryos O/N at 4 °C.

  2. Dehydrate embryos
    1. Remove PFA (collect PFA in waste collecting tube) and wash with PBT three times (use PBT made from DEPC H2O. 400 μl, 10 min each wash, total 3 times).
    2. Equilibrate with methanol to dehydrate the embryos (400 μl, 5 min each wash, three times).
    3. Remove methanol (collect methanol in waste collecting tube).
    4. Add 500 μl to each tube. Store the embryos in at -20 °C (at least 2 h).

      Day 1
  1. Rehydrate embryos
    Wash for 5 min each sequentially in Methanol: PBT (3:1, 1:1, 1:3). (400 μl each wash, use DEPC-PBT.)
    Wash 4x, 5’ each in 100% PBT. (400 μl each wash)
    See note 1 in the end.
    Fix embryos in 4% PFA ( 0.2% glutaradehyde) at RT for 20 min.
    Wash 4x, 5’ each in 100% PBT (400 μl each wash).

  2. Hybridization
    Prehybridize embryos in hybe buffer (300 μl/tube) at 70 °C for 30 min-3 h.
    Replace prehybe with hybe buffer containing the probe(s) of choice. (0.1 ng-1 ng probe/μl hybe buffer)
    Incubate o/n at 70 °C.

    Day 2
    Note: After the RNA probe is hybridized to its template, RNA becomes double stranded and is more stable than single stranded. DEPC-PBT is not necessary.
    1. Remove hybe/probe mixture and store at -20 °C. (can be used up to 3x)
    2. Washes
      75% hybe-/25% 2x SSC         15min, 70 °C
      50% hybe-/50% 2x SSC         15min, 70 °C
      25% hybe-/75% 2x SSC         15min, 70 °C
      100% 2x SSC                         15min, 70 °C
      Wash 2 times in 0.2x SSC      30min, 70 °C
      75% 0.2x SSC/ 25% PBT       10min, RT
      50% 0.2x SSC/ 50% PBT       10min, RT
      25% 0.2x SSC/ 75% PBT       10min, RT
      PBT                                         10min, RT
    3. Add blocking solution to block embryos in at RT for several hours (30 min minimum).

  3. Antibody incubation
    1. After incubation, change buffer for antibody solution (1:5,000 dilution of Roche anti-DIG AP in blocking solution, 500 μl/tube), rock on a platform rotator, at 4 °C O/N.

      Day 3
      DIG staining
    1. Wash quickly in PBT. Use 500 μl/tube.
    2. Wash 6x, 15 min in PBT, shaking at RT. 500 μl/tube.
    3. Wash 1x, 5 min in pre-staining buffer (fresh made). 500 μl/tube. Transfer embryos to 24-well plate use plastic pipets.
    4. Change the buffer to staining buffer (1 ml/well) to the embryos and wrap the plate with aluminum foil and shake at RT.
      1. Staining buffer : Add 4.5 µl of NBT and 3.5 µl BCIP to 1 ml pre-staining buffer.
      2. Check new probes every 30 min to 1 h.
      3. When the staining is done, collect the staining buffer waste. Wash the stained embryos with 1 ml 2x PBT.
      4. Stop reaction by washing in stop solution.
      5. Leave the embryos in stop solution (1 ml/well) or fix in 4% PFA.
    5. Store embryos in stop solution or 4% PFA at 4°C in a closed box.
      Note: For embryos older than 20 somite stage, permeabilization with proteinase K is required to allow the probe to enter the cells.
    6. Incubate in Proteinase K (dilute 1 mg/ml stock 100 fold. 100 μl in 10 ml PBT).
      1. Late somitogenesis (14-22 sec): 2-4 min.
      2. 24 hpf: 10 min.
      3. 36/48 hpf: 20 min.
    7. Wash once (quick) in PBT to get rid of the proteinase K (optional) and continue the fixation.

Recipes

  1. Pronase
    30 mg/ml in E3
  2. 20x SSC (pH7.0)
    NaCl              175.3 g
    NaCitrate       88.2 g
    for 1 L
  3. 10x PBS
    To 800 ml ddH2O dissolve
    NaCl             80 g
    KCl               2 g
    Na2HPO4       14.4 g
    KH2PO4        2.4 g
    pH to 7.4 with HCl and add ddH2O to 1 L.
    * Filter 1x PBS through a 0.2 μm Nalgene filter. Store at RT.
  4. 1x PBT
    10x PBS (pH 7.4) to 1x PBS
    Make a 20% Tween stock. The final concentration of Tween for PBT should be 0.1%.
  5. 4% Paraformaldehyde/PBS
    4 g in 100 ml of PBS, dissolve at 650 C, (or microwave while carefully watching)
  6. Hybe buffer
    50% Formamide                                         25 ml of 100% stock
    5x SSC                                                      12.5 ml of 20x stock
    0.5 mg ml-1 Torula yeast RNA                     1.25 ml of 20 mg/ml stock
    50 mg ml-1 heparin                                     50 µl of 50 mg/ml stock
    0.1% Tween                                              250 µl of 20% Tween
    ddH2O                                                       up to 50 ml
    50 ml total volume
    pH to 6-6.5 with 1 M citric acid ~460 µl.
    For Hybe-, leave out the torula yeast RNA and the heparin.
  7. Heat inactivated Lamb Serum
    Thaw Lamb Serum and heat inactivate at 55 °C for 3 min. Store in aliquots at -20 °C.
  8. Blocking solution
    100 mg BSA
    1 ml 100% Lamb/Sheep Serum
    50 ml PBT
  9. 2x stop solution
    PBS (pH 5.5)
    EDTA 1 mM
  10. Pre-staining buffer
    10 ml 1 M Tris (pH9.5)
    5 ml 1 M MgCl2
    2 ml 5 M NaCl
    500 µl Tween 20
    to 100 ml with water.
  11. Staining buffer
  12. NBT/BCIP
    225 µl 50 mg/ml NBT
    175 µl 50 mg/ml BCIP
    to 50 ml w/ staining buffer

Acknowledgments

This protocol was modified from Reference 1, and developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA. This work was supported by NIH grant R01HD037975.

References

  1. High-resolution in situ hybridization to whole-mount zebrafish embryos. Thisse C1, Thisse B. Nat Protoc. 2008: 3(1):59-69.

材料与试剂

 

1.        BSASigma公司A9418

2.        链霉蛋白酶(罗氏10165921001

3.        PFA(粉末,Fisher

4.        PBTDEPC  H2O))

5.        DEPCSigma公司D5758

6.        甲醇

7.        戊二醛(Sigma公司,G5882

8.        NaCitrate

9.        罗氏抗地高辛AP(罗氏11093274910

10.    NBT / BCIP显色底物(Promega公司,S3771

11.    甲酰胺(高纯级)

12.    EDTA

13.    氯化钠

14.    NaCitrate

15.    氯化钾

16.    磷酸氢二钠

17.    磷酸二氢钾

18.    盐酸

19.    ddH2O

20.    吐温20

21.    圆酵母RNASigma公司,R6225

22.    肝素(Sigma公司,H0777

23.    /羊血清(GibcoBRL16070 096

 

设备

 

1.        杂交孵育器

 

步骤

 

1.        胚胎的准备

1)      使用链霉蛋白酶Dechorinate胚胎。 (添加1滴链霉蛋白酶的溶液到1毫升的E3)。胚胎在室温下保持3-5分钟。 (不要让胚胎在链霉蛋白酶溶液太长!)打破蛋壳吸取胚胎。

2)      收集到1.5 ml管(通常是20个胚胎/每管)的胚胎。去除E3的缓冲液,并用新的1毫升E3缓冲液以去除链霉蛋白酶。去除所有的E3,加入500微升4FA修复的胚胎4°C过夜。

2.        胚胎脱水。

3)      去除PFA(废物收集管收集PFA)与PBT3次。 (使用DEPC H2O配制的PBT400μl,每次洗10分钟,共3次)。

4)      用甲醇平衡脱水的胚胎。 400μl,每洗5分钟,三次)

5)      移除的甲醇(收集甲醇到废物收集管)。每管加入500μl。储存在-20°C(至少2个小时)的胚胎。

1

3.        胚胎的再水化

6)      洗净,甲醇:PBT 3:11:11:3(各400μl洗,DEPC - PBT),每次5分钟。

7)      清洗4x 100PBT(每次400μl洗),每次5秒钟。

8)      在最后附注1

9)      胚胎固定在4PFA(含0.2%戊二醛)在室温下20分钟。

10)  4 x100PBT(每次400μl洗),每次5秒钟。

4.        杂交

11)  Prehybridize胚胎在hybe +缓冲液(300μl/管)70°C30分钟至3小时。

12)  替换hybe prehybe +探针(S)(0.1ng - 1ng探针/μl hybe +缓冲液)

13)  70°C孵育过夜。

2

注:RNA探针杂交后,它的模板,RNA成为双链,比单链稳定。 DEPC – PBT不是必要的。

14)  去除hybe /探针混合物,存储在-20°C(可用于高达3倍)

15)  洗:

75hybe-/25 2X SSC   15' 70 °C

50hybe-/50 2X SSC   15' 70 °C

25hybe-/75 2X SSC   15' 70°C

1002X SSC            15'  70°C

0.2X SSC             30'  70 ° C2

750.2X SSC / 25PBT10'   室温

500.2X SSC / 50PBT10'    室温

250.2X SSC / 75PBT10'    室温

PBT                     10'    室温

16)  添加封闭的溶液(PBT +2%羊血清2 mg / mlBSA),封闭,室温几个小时(至少30分钟)

5.        抗体孵育

17)  孵育后,改变抗体溶液(1:5000稀释的罗氏抗地高辛AP,封闭液配制, 500μl/管),摇动,在4°C过夜。

3

6.        地高辛染色

18)  PBT迅速洗净,500μl/管。

19)  洗净6X15'PBT,在室温下摇晃。 500μl/管。

20)  1x5'预染色缓冲液(新鲜配制)。 500μl/管。使用塑料吸管将胚胎转移到24孔板中。

21)  更改缓冲液的胚胎染色缓冲液(1 ml/孔),用铝箔板包装,室温下摇晃。

a.      染色缓冲液:添加4.5μlNBT3.5μl BCIP 1ml预染色缓冲

b.      每隔30分钟至1小时检查新的探针。

c.      完成染色后,收集染色缓冲液。洗净,用1 ml PBT 2X洗染色胚胎。

d.      添加终止液终止反应(PBS pH5.5EDTA 1 mM2X

e.      胚胎在终止液(1ml/孔)或固定在4%的PFA 

22)  胚胎保存在密闭的终止液或4°C 4PFA

1对于胚胎年龄超过20体节期,Permealization需要蛋白酶K,让探针进入细胞。

23)  在蛋白酶K孵育(稀释1g/ml储存液100x100μl10mlPBT)。

a.      Somitogenesis14 – 22秒):2-4分钟

b.      24 HPF10 分钟

c.      36/48 HPF20分钟

24)  PBT洗一次(快速),以去除蛋白酶K(可选),并继续固定。

 

配方

 

1.        链霉蛋白酶:在E330g/ml

2.        20X SSC

175.3氯化钠

88.2NaCitrate

1L  pH7.0

3.        10X PBS

800ml ddH2O溶解:

80氯化钠

2氯化钾

14.4磷酸氢二钠2.4磷酸二氢钾pH值至7.4,用HCl和添加ddH2O1 L

*通过0.2微米Nalgene过滤的过滤器1X PBS。储存在室温下。

4.        1X PBT

10X PBSpH7.41X PBS。配制20%的吐温储存液。吐温PBT终浓度应为0.1%。

5.        4%多聚甲醛/ PBS

100 ml PBS4,溶解在65°C(或微波,同时仔细观察)

6.        Hybe +溶液:

50%甲酰胺              25ml 100%储存液

5xSSC                  12.5ml20x的储存液

0.5mg/ml的圆酵母RNA   1.25ml20g/ml的储存液

50g/ ml肝素             50μl 50 mg / ml的储存液

0.1%吐温                250μl20%吐温

ddH2O                  50ml

50ml的总体积

pH6-6.51 M柠檬酸 - 460μl

对于Hybe,去除圆酵母RNA和肝素

7.        热灭活羊血清:

解冻羊血清,55°C灭活30秒,分装储存在 - 20 °C

8.        封闭液:

100g BSA 50ml1ml100%的羊/羊血清,50 ml PBT

9.        预染色缓冲液:

10ml1 M Tris pH9.5

5ml 1 M氯化镁

2ml 5 M氯化钠

500μl Tween20

100ml

10.    染色缓冲液+

11.    NBT / BCIP225μl 50 mg / mlNBT175μl 50g/ml BCIP50ml w/染色缓冲液

 

 

English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Jing, L. (2012). In Situ Hybridization (From the Thisse Method). Bio-protocol Bio101: e179. DOI: 10.21769/BioProtoc.179; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册