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[Bio101] pH3 Antibody Staining Protocol: For Zebrafish
[Bio101] 利用组蛋白H3磷酸化抗体检测染色质的状态:​​斑马鱼中   

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Abstract

Phosphorylation of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis. Staining for phosphor histone H3 is a good indicator for the proliferation of the interested cells. This protocol provides a method to stain p-Histone H3 in zebrafish embryos.

Materials and Reagents

  1. PFA
  2. Phosphate buffered saline (PBS)
  3. Acetone 
  4. Alcohol
  5. PBS-Tween 20
  6. H2O2
  7. DMSO
  8. Maleic Acid
  9. p-Histone H3 antibody, 200 μg/ml (Cell Signaling Technology, catalog number: 9701 )
  10. Blocking regent (Roche Diagnostics, catalog number: 1096176 )
  11. Diaminobenzidine (DAB) (Sigma-Aldrich, catalog number: D-5905 )
  12. Peroxidase-conjugated Affinity Goat anti rabbit Ig (Promega Corporation, catalog number: W4011 )
  13. Block solution (see Recipes)
  14. DAB solution  (see Recipes)
  15. 10x MAB buffer  (see Recipes)

Equipment

  1. Shaker
  2. Parafilm
  3. Cold-room

Procedure

  1. DAY 1:
    1. Fix embryos in 4% PFA. Remove PFA before the staining.
    2. 2x PBS (~5 min) rinses to remove remaining PFA. Incubate samples in -20 °C Acetone for 7 min.
    3. Dump acetone (alcohol/acetone waste) and wash samples in water.
    4. Do 2 x 5 min washes with PBST. Place the embryos on shaker at ~ 40 rpm for more effective washing. 
    5. While samples are washing, prepare block solution. Use 500 μl~1 ml/sample.
    6. After 2nd wash, place samples into Block for at least 30 min on the shaker (nonspecific blocking step).
    7. Dilute primary antibody (p-Histone H3 antibody, 200 μg/ml) 1/750 into the Block. (Note: Primary antibody can be reused up to 2 times within the same week!)
    8. Shake samples O/N in the cold-room (at 4 °C). Seal tube with parafilm prevent evaporation of liquid.

  2. DAY 2:
    1. Do 4 x 5 min washes with PBST.
    2. Dilute secondary antibody (Peroxidase-conjugated Affinity Goat anti rabbit Ig) at 1/300 into fresh block.
    3. Shake secondary antibody for 2 h at room temperature (RT).
    4. Remove and discard secondary antibody solution. Do 4 x 15 min washes with PBST.
    5. While samples are washing, prepare DAB solution.
      Note: Combine PBS and two tablets in a 50 ml conical vial. Wrap the mixture in tin foil, as the sample is light sensitive.
    6. Add 24 μl hydrogen peroxide (30%) to DAB mixture (this activates the DAB).
    7. Vortex solution, then transfer to samples (12 μl H2O2 for each tablet=15 ml of DAB mixture).
    8. Remove and discard DAB (special waste stream).
    9. Check if embryos have visible staining.
    10. Wash samples in PBST to remove residual DAB.
    11. Place samples into PFA for storage.
      Note: Once samples are placed into PFA, samples must undergo acetone treatment if restained.

Recipes

  1. Block solution: to make 100 ml of Block
    70 ml of PBST
    20 ml of block reagent (10% in Maleic acid buffer, MAB)
    10 ml of lamb serum
    1 ml of DMSO
  2. 10x MAB buffer (1 L)
    Maleic acid: 116.1 g
    NaCl: 87.66 g
    Water to about 800 ml
  3. Start adding NaOH pellets while stirring the solution. The ppt will not solubilize until the pH reaches about 5.5 to 6.0. At this point, start adding the pellets VERY carefully as it is easy for the pH to overshoot. When the pH reaches 7.5, top up with water to the correct volume and then autoclave if desired.  
  4. DAB solution (developer)
    30 ml of DAB
    30 ml PBS
    2 DAB tablets (toxic)

Acknowledgments

This protocol was modified from the original protocol created and developed in the Len Zon lab at Boston Children’s Hospital and this work was supported by NIH grant R01 HL04880-21.

简介

组蛋白H3的磷酸化与有丝分裂和减数分裂期间的染色体浓缩紧密相关。 荧光蛋白组蛋白H3的染色是感兴趣细胞增殖的良好指标。 这个协议提供了斑马鱼胚胎染色p-组蛋白H3的方法

材料和试剂

  1. PFA
  2. 磷酸盐缓冲盐水(PBS)
  3. 丙酮
  4. PBS-Tween 20
  5. H 2 2 O 2
  6. DMSO
  7. 马来酸
  8. p-Histone H3抗体,200μg/ml(Cell Signaling Technology,目录号:9701)
  9. 封闭试剂(Roche Diagnostics,目录号:1096176)
  10. 二氨基联苯胺(DAB)(Sigma-Aldrich,目录号:D-5905)
  11. 过氧化物酶缀合的亲和性山羊抗兔Ig(Promega Corporation,目录号:W4011)
  12. 块解决方案(参见配方)
  13. DAB解决方案 (参见食谱)
  14. 10x MAB缓冲区 (参见食谱)

设备

  1. 摇床
  2. 石蜡膜
  3. 冷室

程序

  1. 第一天:
    1. 修复胚胎在4%PFA。 在染色前去除PFA。
    2. 2xPBS(〜5分钟)漂洗以除去剩余的PFA。 孵育样品在-20°C丙酮7分钟。
    3. 倾倒丙酮(乙醇/丙酮废物),并在水中洗涤样品。
    4. 用PBST洗涤2×5分钟。 将胚胎放在摇床上以〜40rpm更有效的洗涤。
    5. 在样品洗涤时,制备嵌段溶液。 使用500μl〜1 ml /样品。
    6. 第二次洗涤后,将样品置于摇床上至少30分钟(非特异性阻断步骤)。
    7. 稀释第一抗体(对组蛋白H3抗体,200μg/ml)1/750到块中。 (注意:一抗可在同一周内重复使用2次!)
    8. 在冷室(4°C)摇动样品O/N。 带有石蜡膜的密封管防止液体蒸发
  2. 第2天:
    1. 用PBST洗涤4×5分钟。
    2. 稀释第二抗体(过氧化物酶共轭亲和山羊抗兔Ig)1/300到新鲜块。
    3. 摇动二抗在室温(RT)2小时。
    4. 取出并丢弃二抗溶液。 用PBST洗涤4×15分钟。
    5. 当样品洗涤时,制备DAB溶液 注意:将PBS和两个片剂合并在一个50 ml锥形瓶中。 将混合物包裹在锡箔中,因为样品是光敏的。
    6. 加入24μl过氧化氢(30%)到DAB混合物(这激活DAB)。
    7. 涡旋溶液,然后转移至样品(每个片剂12μlH 2 O 2 2 = 15ml DAB混合物)。
    8. 移除并丢弃DAB(特殊废物流)。
    9. 检查胚胎是否有可见的染色。
    10. 在PBST中洗涤样品以除去残余的DAB。
    11. 将样品放入PFA储存。
      注意:一旦将样品放入PFA中,如果残留,样品必须进行丙酮处理。

食谱

  1. 块溶液:制成100毫升块
    70 ml PBST
    20ml阻断试剂(10%,在马来酸缓冲液,MAB中) 10ml羊乳清蛋白
    1ml的DMSO
  2. 10x MAB缓冲液(1L)
    马来酸:116.1g
    NaCl:87.66g
    水至约800 ml
  3. 在搅拌溶液的同时开始加入NaOH颗粒。 ppt将不溶解,直到pH达到约5.5至6.0。 在这一点上,开始添加丸粒非常仔细,因为它容易pH过度。 当pH达到7.5时,加水至正确的体积,然后如果需要,进行高压灭菌。
  4. DAB解决方案(开发人员)
    30ml DAB
    30 ml PBS
    2 DAB片剂(有毒)

致谢

该协议是从波士顿儿童医院Len Zon实验室创建和开发的原始协议修改的,这项工作由NIH授权R01 HL04880-21支持。

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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jing, L. (2012). pH3 Antibody Staining Protocol: For Zebrafish. Bio-protocol Bio101: e175. DOI: 10.21769/BioProtoc.175;
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suqi zu
USTC
7/30/2013 6:14:41 PM Reply
10/18/2012 1:17:13 AM Reply
Fanglian He
Department of Biology, University of Pennsylvania, USA

As shown in recipe #4, it means 2 DAB (Diamino Bezadine) tablets.

10/18/2012 1:24:03 AM