搜索

Immunostaining Protocol: P-Smad2 (Xenograft and Mice)
免疫染色法:P-Smad2(磷酸化-Smad2,异种移植和小鼠)   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Metastasis depends on a gene program expressed by the tumor microenvironment upon TGF-beta stimulation. CRC (Colorectal cancer) cell lines did not induce robust stromal TGF-beta responses when injected into nude mice as shown by lack of p-SMAD2 accumulation in tumor-associated stromal cells. To enforce high TGF-beta signaling in xenografts, we engineered CRC cell lines to secrete active TGF-beta. Subcutaneous tumors obtained from HT29-M6TGF-β, KM12L4aTGF-β cells and SW48TGF-β cells contained abundant p-SMAD2+ stromal cells.

Materials and Reagents

  1. Paraffin sections (subcutaneous tumors samples or liver metastasis from nude mice respectively injected subcutaneously or intrasplenic with CRC cells)
  2. XILOL
    Note: Xylol also referred to as xylene or dimethylbenzene is a solvent used in histology as a clearing agent to remove paraffin from dried microscope slides prior to staining.
  3. MilliQ H2O
  4. Wash buffer (Dako, catalog number: K800721 )
  5. Rabbit anti-P-Smad2 (Cell Signaling Technology, catalog number: 3108 )
  6. BrightVision poly-HRP anti- Rabbit (Immunologic, catalog number: DPVR-110HRP )
  7. Envision FLEX antibody diluent (Dako, catalog number: K8006 )
  8. Peroxidase Blocking Solution (Dako, catalog number: S202386 )
  9. ImmPACT DAB (Vector Laboratories, catalog number: SK-4105 )
  10. DPX mounting media (Sigma-Aldrich, catalog number: 06522 )
  11. Hematoxylin
  12. Citrate buffer (pH 6) (see Recipes)

Equipment

  1. Oven
  2. Immunostaining apparatus
  3. Autoclave

Procedure

  1. Stove samples at 65 °C just before starting the immunostaining technique. Remove the samples from the oven when the wax present in sections is completely undone.
  2. De-waxing and rehydration: Place slides in a rack to perform following washes (bath).
    1. XILOL: 10 min
    2. XILOL: 10 min
    3. XILOL: 5 min
    4. 100% EtOH: 10 min 
    5. 100% EtOH: 5 min
    6. 96% EtOH: 5 min
    7. 90% EtOH: 10-15 times
    8. 80% EtOH: 10-15 times
    9. 70% EtOH: 10-15 times
    10. 50% EtOH: 10-15 times
    11. 25% EtOH: 10-15 times
    12. H2O MilliQ: 10-15 times
  3. Antigen retrieval.
    1. Citrate Buffer (pH 6)
    2. Time: 20 min autoclave (121 °C)
  4. 3 washes 5 min with 1 ml 1x wash buffer.
  5. Blocking endogenous peroxidase.
    1.  200 ml Peroxidase Blocking Solution
    2. Time: 10 min
  6. 3 washes 5 min with 1ml 1x wash buffer.
  7. Incubation with primary antibody.
    1. Antibody: Rabbit anti-P-Smad2
    2. Dilution 1/200 in Envision FLEX antibody diluent
    3. 200 µl/sample
    4. O/N 4 °C
  8. 3 washes 5 min with 1 ml 1x wash buffer.
  9. Incubation with antibody BrightVision.
    1.  Antiboby: BrightVision anti-Rabbit
    2. 150 µl/sample
    3. Time: 45 min at room temperature
  10. 3 washes 5 min with 1 ml 1x wash buffer.
  11. Revealed with ImmPACT DAB.
    1.  200 µl/sample
    2. Time: 10 min
  12. 3 washes 5 min with distilled water.
  13. Hematoxilin (1 ml) counterstaining, time: 2 min.
  14. Rinse in distilled water bath.
  15. Rinse in TAP water bath.
  16. Dehydration and mounting with DPX.

Representative data



Figure 1. p-SMAD2 staining (arrowhead) in liver metastasis generated after intrasplenic injection of CRC cells. E: epithelial cells, Str: stromal cells. Scale bars = 10 μm

Recipes

  1. Citrate buffer (pH 6)
    Citrate 5.8 g sodium citrate
    Set pH 6 with citric acid
    2 L MilliQ

Acknowledgments

A.C. and D.V.F.T hold a Juan de la Cierva postdoctoral fellowship, E.E. an FPI PhD fellowship (both from Spanish Ministry of Science and Innovation). This work has been supported by grants from Instituto de Salud Carlos III FEDER (RD09/0076/00036) and the “Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC), to E.B. from the European Research Council (Starting grant - 208488) and Consolider programmes (MICINN), to E.S. and A.C. by the Spanish Ministry of Science and Innovation, to JM by NIH grant CA34610 and to RM by grants PS09/00965 (MICINN) and NanoCoMets (CIBERBBN).

References

  1. Calon, A., Espinet, E., Palomo-Ponce, S., Tauriello, D. V., Iglesias, M., Cespedes, M. V., Sevillano, M., Nadal, C., Jung, P., Zhang, X. H., Byrom, D., Riera, A., Rossell, D., Mangues, R., Massague, J., Sancho, E. and Batlle, E. (2012). Dependency of colorectal cancer on a TGF-beta-driven program in stromal cells for metastasis initiation. Cancer Cell 22(5): 571-584.

简介

转移依赖于在TGF-β刺激时由肿瘤微环境表达的基因程序。 当注射到裸鼠中时,CRC(结肠直肠癌)细胞系不诱导强烈的基质TGF-β应答,如肿瘤相关基质细胞中缺乏p-SMAD2积累所示。 为了在异种移植物中强化高TGF-β信号传导,我们设计CRC细胞系以分泌活性TGF-β。 从HT29-M6TGF-β,KM12L4aTGF-β细胞和SW48TGF-β细胞获得的皮下肿瘤含有丰富的p-SMAD2 + 基质细胞。

材料和试剂

  1. 石蜡切片(皮下肿瘤样品或分别从皮下或脾内注射CRC细胞的裸鼠的肝转移)
  2. XILOL
    注意:二甲苯也称为二甲苯或二甲苯是在组织学中用作清除剂的溶剂,以在染色前从干燥的显微镜载玻片上除去石蜡。
  3. MilliQ H sub 2 O 2 /
  4. 洗涤缓冲液(Dako,目录号:K800721)
  5. 兔抗P-Smad2(Cell Signaling Technology,目录号:3108)
  6. BrightVision poly-HRP抗兔(Immunologic,目录号:DPVR-110HRP)
  7. Envision FLEX抗体稀释剂(Dako,目录号:K8006)
  8. 过氧化物酶封闭溶液(Dako,目录号:S202386)
  9. ImmPACT DAB(Vector Laboratories,目录号:SK-4105)
  10. DPX安装介质(Sigma-Aldrich,目录号:06522)
  11. 苏木精
  12. 柠檬酸盐缓冲液(pH 6)(参见配方)

设备

  1. 烤箱
  2. 免疫染色仪
  3. 高压灭菌器

程序

  1. 在开始免疫染色技术之前在65℃下烘烤样品。 当存在于切片中的蜡完全去除时,从烘箱中取出样品
  2. 脱蜡和再水合:将玻片放在架子上进行以下洗涤(浴)。
    1. XILOL:10分钟
    2. XILOL:10分钟
    3. XILOL:5分钟
    4. 100%EtOH:10分钟
    5. 100%EtOH:5分钟
    6. 96%EtOH:5min
    7. 90%EtOH:10-15倍
    8. 80%EtOH:10-15倍
    9. 70%EtOH:10-15倍
    10. 50%EtOH:10-15倍
    11. 25%EtOH:10-15倍
    12. H 2 O MilliQ:10-15次
  3. 抗原检索。
    1. 柠檬酸盐缓冲液(pH 6)
    2. 时间:20分钟高压釜(121℃)
  4. 3次用1ml 1x洗涤缓冲液洗涤5分钟
  5. 阻断内源性过氧化物酶。
    1.   200 ml过氧化物酶封闭溶液
    2. 时间:10分钟
  6. 3次用1ml 1x洗涤缓冲液洗涤5分钟
  7. 与第一抗体孵育。
    1. 抗体:兔抗P-Smad2
    2. 稀释1/200在Envision FLEX抗体稀释剂
    3. 200μl/样品
    4. O/N 4℃
  8. 3用1ml 1x洗涤缓冲液洗涤5分钟。
  9. 与抗体BrightVision孵育。
    1.   Antiboby:BrightVision anti-Rabbit
    2. 150μl/样品
    3. 时间:室温下45分钟
  10. 3用1ml 1x洗涤缓冲液洗涤5分钟。
  11. 揭示了ImmPACT DAB。
    1.   200μl/sample
    2. 时间:10分钟
  12. 3用蒸馏水洗涤5分钟
  13. 苏木素(1ml)复染色,时间:2分钟。
  14. 用蒸馏水冲洗。
  15. 在TAP水浴中冲洗。
  16. 用DPX脱水和安装。

代表数据



图1.在脾细胞注射CRC细胞后产生的肝转移中的p-SMAD2染色(箭头)。E:上皮细胞,Str:基质细胞。 比例尺=10μm

食谱

  1. 柠檬酸缓冲液(pH 6)
    柠檬酸盐5.8g柠檬酸钠
    用柠檬酸调节pH 6 2 L MilliQ

致谢

A.C.和D.V.F.T拥有Juan de la Cierva博士后研究金,E.E.一名FPI博士研究生奖学金(均来自西班牙科学和创新部)。 这项工作得到了Salud Carlos III FEDER研究所(RD09/0076/00036)和"加拿大肿瘤学院院长赞助的肿瘤研究"(XBTC)给予E.B.的资助。 从欧洲研究理事会(启动补助金 - 208488)和Consolider计划(MICINN)到E.S. 由西班牙科学和创新部授予JM,NIH授予JM,CA34610授予JM,通过授予PS09/00965(MICINN)和NanoCoMets(CIBERBBN)授予RM。

参考文献

  1. Calon,A.,Espinet,E.,Palomo-Ponce,S.,Tauriello,DV,Iglesias,M.,Cespedes,MV,Sevillano,M.,Nadal,C.,Jung,P.,Zhang,XH,Byrom D.,Riera,A.,Rossell,D.,Mangues,R.,Massague,J.,Sancho,E。和Batlle,E。(2012)。 结肠直肠癌对基质细胞中TGF-β驱动程序对转移起始的依赖性。 a> Cancer Cell 22(5):571-584。
  • 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。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Calon, A., Espinet, E., Palomo-Ponce, S., Tauriello, D. V., Iglesias, M., Céspedes, M. V., Sevillano, M., Nadal, C., Jung, P., Zhang, X. H., Byrom, D., Riera, A., Rossell, D., Mangues, R., Massague, J., Sancho, E. and Batlle, E. (2014). Immunostaining Protocol: P-Smad2 (Xenograft and Mice). Bio-protocol 4(9): e1119. DOI: 10.21769/BioProtoc.1119.
提问与回复

(提问前,请先登录)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。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。