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Double in situ hybridization I very useful to examine the relationship between the expression of two genes. But it is tricky because of the cross reaction of two different antibodies. This protocol is a valid method to do double color in situ hybridization in zebrafish embryos.

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[Bio101] Double In Situ Hybridization (the Fish Method)
[Bio101] 双色荧光原位杂交技术(FISH)

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 Pennsylvani, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
2/5/2012, 7567 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.178

[Abstract] Double in situ hybridization I very useful to examine the relationship between the expression of two genes. But it is tricky because of the cross reaction of two different antibodies. This protocol is a valid method to do double color in situ hybridization in zebrafish embryos.

[Abstract] 双倍原位杂交。我的原位杂交研究的两个基因的表达之间的关系非常有用。但它是非常棘手,因为两个不同的抗体交叉反应。在斑马鱼胚胎进行双色原位杂交技术一个有效的方法。

Materials and Reagents

  1. Methanol
  2. 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP)
  3. Nitrotetrazolium blue chloride (NBT)
  4. EDTA
  5. INT
  6. Tris
  7. Tween 20
  8. Glycine
  9. Na2HPO4
  10. HCl
  11. NaCl
  12. Sodium Citrate
  13. MgCl2
  14. KCl
  15. PFA
  16. Sheep serum
  17. BSA
  18. Citric acid
  19. Formamide
  20. Dechorionate
  21. Hybe+ buffer (5ml/tube)
  22. Torula Yeast RNA (Sigma-Aldrich, catalog number: R6225)
  23. Heparin (Sigma-Aldrich, catalog number: H0777 )
  24. Lamb Serum (GibcoBRL, catalog number: 16070096 )
  25. Fast Red staining buffer (FRSB) [1 M Tris (pH 8.2), 0.1% Tween]
  26. Proteinase K (Roche Diagnostics, catalog number: 10165921001 )
  27. Fast Red talets (Roche Diagnostics, catalog number: 11496549001 ) or INT/BCIP (Roche Diagnostics, catalog number: 11681460001 )
  28. NBT/BCIP (Promega Corporation, catalog number: S3771 )
  29. Anti-DIG -AP (Roche Diagnostics, catalog number: 11093274910 )
  30. Anti-Fluorescine AP (Roche Diagnostics, catalog number: 11426338910 )
  31. NBT/BCIP (see Recipes)
  32. 1x PBT (see Recipes)
  33. 20x SSC (see Recipes)
  34. 10x PBS (see Recipes)
  35. 4% Paraformaldehyde (see Recipes)
  36. Hybe+ buffer (5ml/tube) (see Recipes)
  37. Heat Inactivated Lamb Serum (see Recipes)
  38. Blocking solution (see Recipes)
  39. Staining buffer (see Recipes)
  40. Stop solution (see Recipes)

Equipment

  1. Rotator
  2. Nalgene filter
  3. Hybridization Incubator

Procedure

  1. Preparation of Embryos
    1. Fix in p-formaldehyde (4%) o/n at 4 °C.
    2. Wash twice in PBT (fresh made stock) and dechorionate.
    3. Wash and equilibrate with methanol (3x, for 5 min each).
    4. Store at -20 °C.

      Day 1
  2. Rehydration of Embryos
    1. Wash for 5 min each in Methanol: PBT sequentially (3:1, 1:1, 1:3).
    2. Wash 4x, 5 min each in 100% PBT.
    3. Incubate in Proteinase K (dilute 1 mg/ml stock 100 fold. 100 μl in 10 ml PBT).
      1. Younger than “bud”: 30 sec.
      2. Early Somitogenesis: 1-2 min.
      3. Late Somitogenesis (14-22 sec): 2-4 min.
      4. 24 hpf: 10 min.
      5. 36/48 hpf: 20 min.
    4. Wash once (quick) in PBT to get rid of the proteinase K. (optional).
    5. Refix for 20 min in 4% p-formaldehyde at room temperature (RT).
    6. Rinse 5x, 5 min in PBT.

  3. Hybridization
    1. Prehybridize embryos in hybe+ buffer (5 ml/tube) at 70 °C for 2-5 h.
    2. Replace prehybe with hybe+ buffer containing the two probes of choice (~150-200 ng of each probe/200 μl hybe+ buffer).
    3. Incubate o/n at 70 °C.

      Day 2
    4. Remove hybe/probe mixture and store at -20 °C (can be used up to 3x).
    5. Washes:
      1. 100% prewarmed hybe- buffer, 10 min, 70 °C.
      2. 75% hybe-/25% 2x SSC, 15 min, 70 °C.
      3. 50% hybe-/50% 2x SSC, 15 min, 70 °C.
      4. 100% 2x SSC, 15 min, 70 °C.
      5. Wash 2 times in 0.2x SSC, 30 min, 70 °C.
      6. 75% 0.2x SSC/ 25% PBT, 10 min, RT.
      7. 50% 0.2x SSC/ 50% PBT, 10 min, RT.
      8. 25% 0.2x SSC/ 75% PBT, 10 min, RT.
      9. PBT, 10 min, RT.
    6. Block embryos in PBT/2% sheep serum/2 mg/ml BSA at RT for 2 h.

  4. First Ab Incubation (anti-fluorescein-AP)
    1. Incubate embryos with 500 μl of antibody solution (1:2,000 dilution) for 2 h at RT or o/n at 4 °C, rocking on a rotator.

  5. Staining the embryos (Fast red method)
    1. Wash excess ab off embryos 6x, 15 min in PBT, shaking at RT.
    2. Wash 2-3x in FRSB (1 M Tris, pH 8.2, 0.1% Tween).
    3. Stain in Fash Red Solution (1 tablet in 2 ml FRSB).
    4. After staining is complete wash 3x, 5 min each at RT in 0.1 M glycine (pH 2.2), 0.1% tween to remove the antibody.
    5. Wash 3-4x in PBT to remove all the glycine.
      Or staining the embryos (INT method)
    1. Wash embryos 2x for 5 min each in staining buffer.
    2. Stain embryos in the following solution: 31.5 μl INT, 35 μl BCIP to 10 ml with staining buffer.
    3. To stop reaction fix for 20 min at RT in 4% PFA.
    4. To get rid of primary ab, wash 3x for 5 min each at RT in 0.1 M glycine (pH 2.2), 0.1% tween 20.
    5. Wash 3x for 15 min each at RT in PBT to remove all the glycine.

  6. Second Ab Incubation (anti-DIG-AP)
    1. Incubate embryos with 500 μl of antibody solution (1:5,000 dilution) o/n, rocking on anutator, at 4 °C or for 2 h at RT.

      Day 3
  7. DIG Staining
    1. Wash quickly in PBT.
    2. Wash 6x, 15 min in PBT, shaking at RT.
    3. Wash 2-3x, 5 min in staining buffer.
    4. Add 90 μl of 50 mg/ml NBT and 70 μl of 50 mg/ml BCIP to 20 ml staining buffer.
    5. Add about 500 ml of staining buffer to embryos and wrap rack in aluminum foil and shake at RT. Check new probes every 30 min to 1 h.
    6. Stop reaction by washing in Stop Solution 3x [PBS (pH 5.5), EDTA 1 mM)] or 4% PFA.
    7. Store embryos in 4% PFA at 4 °C in a closed box.

Recipes

  1. 20x SSC (pH 7.0)
    NaCl            175.3 g
    NaCitrate     88.2 g
    for 1 L
  2. 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 .2 μm Nalgene filter. Store at RT.
  3. 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%.
  4. 4% Paraformaldehyde/PBS
    4 g in 100 ml of PBS, dissolve at 650 C, (or microwave while carefully watching).
  5. Hybe+ buffer
    50% Formamide 25 ml of 100% stock
    5x SSC 12.5 ml of 20x stock
    0.5 mg/ml Torula Yeast RNA 1.25 ml of 20 mg/ml stock
    50 mg/ml heparin 50 μl of 50 mg/ml stock
    0.1% Tween 250 μl of 20% Tween
    1 M citric acid 460 μl
    50 ml total volume
  6. 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 30 min. Store in aliquots at -20 °C.
  8. Blocking solution
    100 mg BSA (Sigma-Aldrich)
    1 ml 100% Lamb/Sheep Serum
    50 ml PBT
  9. Staining buffer
    10 ml 1 M Tris (pH 9.5)
    5 ml 1 M MgCl2
    2 ml 5 M NaCl
    500 μl Tween 20
    to 100 ml with water
  10. NBT/BCIP
    225 μl 50 mg/ml NBT
    175 μl 50 mg/ml BCIP
    to 50 ml staining buffer
  11. 3x stop solution
    PBS (pH 5.5)
    EDTA 1 mM
  12. Pre-Staining Buffer
    10 ml 1 M Tris (pH 9.5)
    5 ml 1 M MgCl2
    2 ml 5 M NaCl
    500 μl Tween 20
    To 100 ml with water
  13. Satining buffer
  14. 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 the original protocol developed in the Len Zon lab at Boston Children’s Hospital, Boston, USA and supported by NIH grant R01 HL04880-21.

材料与试剂

 

1.        甲醇

2.        蛋白酶KRoche公司10165921001

3.        快速红taletsRoche公司11496549001)或INT / BCIPRoche公司11681460001

4.        Tris

5.        吐温

6.        甘氨酸

7.        NBT / BCIPPromega公司,S3771

8.        - DIG - APRoche 公司 11093274910

9.        -Fluorescine APRoche1公司1426338910

10.    EDTA

11.    PBS

12.    PFA 

13.    氯化钠

14.    NaCitrate

15.    氯化钾

16.    磷酸氢二钠

17.    盐酸

18.    甲酰胺

19.    串酵母菌RNASigma公司,R6225

20.    肝素(Sigma公司,H0777

21.    柠檬酸

22.    羊血清(GibcoBRL16070 096

23.    氯化镁

 

设备

 

1.        旋转器

2.        杂交孵育器

 

步骤

 

1.        胚胎的预处理

1)      P -甲醛过夜(4%)4 °C固定。

2)      PBT洗两次(新鲜的储存液配制)和dechorionate

3)      清洗和用甲醇平衡(每5分钟1次,3次)

4)      储存在-20°C

1

2.        补液的胚胎

1)      在甲醇:PBT  3:11:11:3洗涤,每次5分钟,

2)      每次5 min100PBT洗涤,4次,

3)      在蛋白酶K孵育(稀释1 mg/ml 储存液100倍。100μl 稀释在10 ml PBT的)。

a.      幼龄期“bud”30

b.      早期Somitogenesis1-2分钟

c.      后期Somitogenesis14 - 22 s):2-4分钟

d.      24 hpf10 分钟

e.      36/48 hpf20分钟

4)      PBT洗一次(快速),以去除蛋白酶K(可选)

5)      4%的P -甲醛在室温下重固定20分钟。

6)      PBT 5分钟,洗涤5次,

3.        杂交

1)      Prehybridize胚胎置于hybe +缓冲液(.5ml/tube),70°C2-5小时。

2)      22.更换prehybe hybe ++缓冲液包含已经选择好的两个探针(?150-200 ng/每管/200 μl hybe+缓冲区液)的缓冲区。

3)      70°C孵育。

2

4)      清除hybe /探针混合物,存储在-20°C和(可用于3x

5)      洗涤:

a.      100%预热Hybe缓冲70°C 10分钟

b.      75hybe-/252x SSC70°C 15分钟?

c.      50hybe-/502x SSC 70 °C 15分钟

d.      25hybe-/752x SSC70°C 15分钟

e.      1000.2x SSC 70 ° C 15分钟

f.        0.2x SSC 70℃30分钟,清洗2

g.      0.2x SSC75/ 25PBT ,室温10分钟

h.      0.2x SSC50/ 50PBT ,室温10分钟

i.         250.2x SSC / 75PBT ,室温10分钟

j.         PBT 室温10分钟

6)      封闭胚胎在PBT / 2%羊血清/ 2 mg / mlBSA室温下2小时

4.        第一抗体孵化(抗荧光素- AP

1)      500μL抗体溶液(1:2000稀释)孵育胚胎为2小时,在室温或4 °C过夜,在旋转器摇晃。

5.        胚胎染色(FAST红法)

1)      PBT 15分钟洗掉胚胎6x多余的AB,在室温下摇晃。

2)      用快速红染色缓冲液(FRSB)洗涤2 - 3x ,(1M Tris, pH 8.2, 0.1% 吐温

3)      FASH溶液中染色(1   2 ml FRSB

4)      染色后洗3次,室温中5分钟。

5)      50.1M甘氨酸,pH 2.2 0.1吐温,以洗去抗体。

6)      6PBT洗涤 3 – 4次,洗去所有的甘氨酸。

5.        胚胎染色(诠释方法)

1)      在染色缓冲液中,洗涤胚胎5分钟,2

2)      在下述溶液中染色胚胎:31.5 μl INT, 35 μl BCIP 用染色缓冲液稀释到10 mls

3)      停止反应,在在室温下固定于4%的多聚甲醛

4)      为了清除抗的影响,在室温下用0.1M甘氨酸,pH 2.20.1tween20洗涤。每次5分钟,共3次。

5)      PBT室温洗涤 3 – 4次,洗去所有的甘氨酸。

6.        第二抗体孵化(抗- DIG - AP

1)      500μl抗体溶液(1:5000稀释)过夜孵育,在旋转器内摇动,在4℃过夜或室温下2小时。

3

7.        地高辛染色

1)      PBT速洗

2)      洗涤6次,PBT 15分钟,在室温下摇动

3)      32 – 3次,染色缓冲液5分钟洗涤

4)      4)加入90μl 50 mg / mlNBT70μl50 mg/ml20 ml的染色缓冲液BCIP

5)      5)加入约500 ml染色缓冲液到胚胎,然后铝箔包裹,并在室温下摇动。每隔30分钟至1小时检查新的探针。通过终止液(PBS pH5.5EDTA 1 mM)的3倍或4??%多聚甲醛终止反应

6)      6)用4??%多聚甲醛固定胚胎在一个封闭的盒子。

 

配方

 

1.        20x SSC

175.3氯化钠

88.2NaCitrate

1 L pH7.02.

2.        10x PBS

800 ml 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和加ddH2O1 L

*通过0.2微米Nalgene过滤器过滤1x PBS。储存在室温下。

3.        1x PBT

10x PBSpH7.41x PBS。使20%的吐温股票。PBT中的吐温终浓度应为0.1%。

4.        4%多聚甲醛/ PBS

4100mlPBS溶解在650 C(或微波,同时仔细观察)

5.        Hybe +溶液:

50%甲酰胺25ml100%储存液

5 x SSC12.5ml 20 x的储存液

0.5 mg/ml酵母RNA 1.25ml 20 mg/ml的储存液

50 mg / ml肝素50μL50 mg / ml的储存液

0.1%吐温250μl 20%吐温

1 M柠檬酸 460 μl

50ml的总量

6.        对于Hybe,去除了酵母RNA和肝素

7.        热灭活羊血清:

解冻羊血清(GibcoBRL16070 096)和加热在55°C 30分钟灭活。储存分装在 - 20°C

8.        封闭液:

100 mg BSASigma公司),

1ml 100%的羊/羊血清,

50ml PBT

9.        抗地高辛抗体:Roche1093 247

10.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

11. NBT / BCIP

225μl 50 mg /mlNBT

175μl 50 mg /ml BCIP

50ml w/ 染色缓冲液

 

 

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How to cite this protocol: Jing, L. (2012). Double In Situ Hybridization (the Fish Method). Bio-protocol Bio101: e178. DOI: 10.21769/BioProtoc.178; Full Text



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