参见作者原研究论文

本实验方案简略版
Jul 2017

本文章节


 

Protocol for in situ Proximity Ligation Assay (PLA) and Microscopy Analysis of Epidermal Growth Factor Receptor (EGFR) Homodimerization
原位邻位连接检测和显微镜分析表皮生长因子的同源二聚化的实验方法   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Oncogenic drivers play central roles in tumorigenesis as well as in tumor cell survival and proliferation. Mutations of the epidermal growth factor receptor gene (EGFR) that result in constitutive activation of the receptor tyrosine kinase have been identified as oncogenic drivers in a subset of non-small cell lung cancer (NSCLC). PCR-based assays are usually adopted for the detection of EGFR mutations, but no methods to detect EGFR activation that are not based on mutation identification have been established in the clinical setting. We describe a proximity ligation assay (PLA) used to visualize and quantitate EGFR homodimerization in NSCLC cell lines and tissue specimens. Rabbit monoclonal antibodies against EGFR were conjugated to PLUS or MINUS PLA oligonucleotide arms using Probemaker. Annealing of the PLUS and MINUS PLA probes occurred when two EGFR monomers were in close proximity, and repeat sequences in the annealed oligonucleotide complexes were amplified then recognized by a fluorescently-labeled oligonucleotide probe. PLA signals were detected and counted with a fluorescence microscope. We demonstrate the detection of EGFR homodimers by PLA analysis in a quantitative manner in both NSCLC cell lines and tissue samples obtained by transbronchial lung biopsy. PLA methods are a new tool for the detection and quantitation of protein-protein interactions such as homodimers, heterodimers, and fusion proteins.

Keywords: Oncogenic driver (致癌驱动), Epidermal growth factor receptor (EGFR) (表皮生长因子), Anaplastic lymphoma kinase (ALK) (间变性淋巴瘤激酶), Non-small cell lung cancer (NSCLC) (非小细胞肺癌), Proximity ligation assay (PLA) (邻位连接检测), Dimerization (二聚化)

Background

Activating mutations of the epidermal growth factor receptor gene (EGFR) are currently detected by PCR-based assays (Nagai et al., 2005). However, the visual detection and quantitation of activated EGFR in the clinical setting has not been established. In situ proximity ligation assay (PLA) is a technology that uses Duolink® In situ reagents (see References 1 and 2) to create probes by conjugating oligonucleotides to antibodies. When two different types of PLA probes (PLUS and MINUS) are in close proximity (40 nm), annealing occurs which generates an amplified circular DNA. The signal from each detected pair of PLA probes is visualized as an individual spot, and the number of PLA signals per cell can be counted with a fluorescence microscope (Figure 1).

The PLA method can be used to detect any protein-protein interactions in close proximity. We have now applied the PLA using primary antibodies derived from the same species to visualize and quantitate EGFR homodimerization. Furthermore, we detected the formation of EGFR-human epidermal growth factor receptor2 heterodimers and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion protein in NSCLC cell lines (Ota et al., 2017). This method will be applicable to the detection of other dimerizations and fusions.


Figure 1. In situ PLA principle for EGFR homodimer. A. PLA probes, created by conjugating PLA oligonucleotides and monoclonal antibodies for EGFR, bind to EGFR. B. Connected oligonucleotides hybridize and create multiple circular DNA molecules. C. Fluorescently-labeled detection of oligonucleotides hybridizing to the DNA circle. (Pictures were taken from the Duolink® In situ User Guide [References 1 and 2]).

Materials and Reagents

  1. Standard pipette tips with a volume capacity of 10 μl, 20 μl, 100 μl, 200 μl, and 1,000 μl (Thermo Fisher Scientific, catalog numbers: 2140, 2149P, ART 10REACH, ART 20P)
  2. Kimwipe waste paper sheet S-200 (Crecia, catalog number: 62011)
  3. 12-mm-diameter uncoated cover glasses (Matsunami Glass, catalog number: C012001)
  4. 24-well plates (Greiner Bio-One, cell culture multiwell plates, catalog number: 66)
  5. Staining jar (Matsunami, catalog number: No. 13 BT500)
  6. Dako Pen (Daido Sangyo, PAP-SPAP Pen Super-Liquid Blocker)
  7. Serological pipettes
  8. Slide glasses
  9. NSCLC cell lines: H1975 cells
  10. Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Gibco, catalog number: 61870-036)
  11. Dulbecco's modified Eagle’s medium (DMEM) (Invitrogen, Gibco, catalog number: 12634010)
  12. Fetal bovine serum (FBS) (Sigma, catalog number: 14C438FBS)
  13. Penicillin/Streptomycin solution (Invitrogen, catalog number: 15140122)
  14. Phosphate-buffered saline (PBS) (LSI, catalog number: RM102-PN)
  15. 4% paraformaldehyde in PBS (Wako, catalog number: 16320145)
  16. ImmunoSaver (Wako, catalog number: 9706192)
  17. Duolink® In situ oligonucleotide PLUS (-20 °C) (Sigma-Aldrich, catalog number: DUO92009)
  18. Duolink® In situ oligonucleotide MINUS (-20 °C) (Sigma-Aldrich, catalog number: DUO92010)
  19. Conjugation buffer (-20 °C) (Sigma-Aldrich, catalog number: DUO92009)
  20. Stop reagent (-20 °C) (Sigma-Aldrich, catalog number: DUO92009)
  21. Storage solution (-20 °C) (Sigma-Aldrich, catalog number: DUO92009)
  22. 20x assay reagent (-20 °C) (Sigma-Aldrich, catalog number: DUO92009)
  23. Blocking solution (4 °C) (Sigma-Aldrich, catalog number: DUO92009)
  24. PLA probe diluent (4 °C) (Sigma-Aldrich, catalog number: DUO92009)
  25. Rabbit monoclonal antibodies to EGFR (Abcam, catalog number: ab52894)
  26. 5x Ligation buffer (-20 °C) (Sigma-Aldrich, catalog number: DUO92008)
  27. 1x Ligase (-20 °C) (Sigma-Aldrich, catalog number: DUO92008)
  28. 5x Amplification Red (-20 °C) (Sigma-Aldrich, catalog number: DUO92008)
  29. 1x Polymerase (-20 °C) (Sigma-Aldrich, catalog number: DUO92008)
  30. Duolink® In situ mounting medium with DAPI (Sigma-Aldrich, catalog number: DUO82040)
  31. 10% neutral-buffered formalin
  32. Paraffin
  33. Xylene
  34. Ethanol solutions
  35. Nail polish
  36. Culture media (10% FBS) (see Recipes)
  37. Wash buffer A (Sigma-Aldrich, catalog number: DUO82049, see Recipes)
  38. Wash buffer B (Sigma-Aldrich, catalog number: DUO82049, see Recipes)

Equipment

  1. Manual pipettes: set of 10 μl, 20 μl, 100 μl, 200 μl, and 1,000 μl (Mettler Toledo, catalog number: Pipet-Lite XLS+ 17014409, 17014412, 17014408, 17014411, 17014407)
  2. Tweezers
  3. Freezer
  4. Direct-Q® 5 UV Remote Water Purification System (Merck, model: Direct-Q® UV 5 Remote, catalog number: ZRQSVR5WW)
  5. Autoclave (Tomy, catalog number: LSX-500)
  6. Vortex mixer (Scientific Industries, model: Vortex-Genie 2, catalog number: S1-0286)
  7. Humidity chamber (Incubation Chamber, Cosmo Bio, catalog number: 10DO)
  8. Freeze block for enzymes (-20 °C) (Eppendorf, catalog number: 3880001018)
  9. Fluorescence microscope (Keyence, model: BZ-8100)

Software

  1. BZ Analyzer software (Keyence)
  2. Excel (Microsoft)
  3. GraphPad Prism 5.0 (GraphPad Software)

Procedure

Note: All procedures are performed for four samples.

  1. Preparation of the cells (Figure 2)
    1. Place 12-mm-diameter cover glasses in each well of a 24-well plate.
    2. Grow the cells to 60% confluence on the cover glasses in 500 μl of the recommended culture media at 37 °C, with 5% CO2.
    3. Remove media from the wells and wash twice each with 1 ml PBS. 
    4. Add 500 μl 4% paraformaldehyde for 15 min at room temperature.
    5. Remove the solution from the well and wash twice each with 1 ml PBS.
    6. Take the cover glasses from the wells using tweezers.


    Figure 2. Preparation for PLA method with cell lines. A. Cover glasses in each well of the plate (left). Cells grown on cover glasses in the culture media (right). B. Post-PLA slide glasses mounted with cover glasses using DAPI and sealed with nail polish.

  2. Preparation of the tumor specimens
    1. Fix surgical specimens in 10% neutral-buffered formalin, embed them in paraffin, and section at a thickness of 4 μm.
    2. Remove paraffin with xylene, rehydrate with a graded series of ethanol solutions, and wash twice with PBS.
    3. Add 300 μl ImmunoSaver to 60 ml Milli-Q water in the staining jar.
    4. Autoclave the slides in the staining jar for 45 min at 98 °C and incubate at room temperature.
    5. Wash the slides twice with PBS.
    6. Draw a circle around the tumor specimen of around 1 cm2 with the Dako Pen.

  3. Conjugation of the PLA oligonucleotide to the antibody
    1. Thaw conjugation buffer at room temperature and vortex.
    2. Add 2.1 μl conjugation buffer to 21 μl rabbit monoclonal anti-EGFR antibody and mix gently with a pipette.
    3. Transfer the antibody solution from Step C2 to one vial of oligonucleotide PLUS or oligonucleotide MINUS and mix gently with a pipette.
    4. Incubate at room temperature overnight.
    5. Vortex stop reagent solution and add 2.1 μl to the solution from Step C3 and incubate at room temperature for 30 min.
    6. Vortex storage solution and add 25.2 μl to the solution prepared in Step C5 and store at 4 °C.
    7. The stored solution can be used within one month.

  4. Blocking
    1. Vortex blocking solution and transfer 40 μl to each sample.
    2. Put the slides in a pre-heated humidity chamber and incubate in a humidified 37 °C, 5% CO2 incubator for 30 min.

  5. PLA probes
    1. Dilute the conjugated antibodies from Step C6 in the PLA probe diluent.
      1. Prepare 200 μl of 1x working assay reagent by mixing 10 μl of 20x stock solution and 190 μl of PLA probe diluent.
      2. Add 8 μl PLUS or MINUS antibody solution (Step C6) to 72 μl working assay reagent (from Step E1a) and incubate for 20 min at room temperature.
    2. Gently remove the blocking solution from the samples using a Kimwipe waste paper sheet without touching the cells.
    3. Add 38 μl (19 μl PLUS probe and 19 μl MINUS probe) in total diluted PLA probes PLUS solution and MINUS solution to each sample.
    4. Mix gently with a pipette.
    5. Incubate in a humidity chamber for 2 h at room temperature.

  6. Ligation
    1. Dilute 32 μl ligation buffer (5x) in 124 μl Milli-Q water and mix.
    2. Gently remove the PLA probe solution from the samples using a Kimwipe waste paper sheet without touching the cells.
    3. Gently wash the slides twice for 5 min each in 500 μl Wash Buffer A.
    4. Add 4 μl ligase to 156 μl ligation buffer (1x) and vortex.
    5. Add 39 μl solution to each sample.
    6. Put the slides in a pre-heated humidity chamber and incubate in a humidified 37 °C, 5% CO2 incubator for 30 min.

  7. Amplification
    1. Dilute 32 μl amplification red (5x) in 126 μl Milli-Q water and mix.
    2. Gently remove the ligation solution from the samples using a Kimwipe waste paper sheet without touching the cells.
    3. Gently wash the slides twice for 2 min each in 500 μl Wash Buffer A.
    4. Add 2 μl polymerase solution to 158 μl amplification red (1x) and vortex.
    5. Add 39 μl solution to each sample.
    6. Put the slides in a pre-heated humidity chamber and incubate with a humidified 37 °C, 5% CO2 incubator for 30 min.

  8. Preparation for imaging
    1. Gently remove the amplification-polymerase solution from the samples using a Kimwipe waste paper sheet without touching the cells.
    2. Gently wash the slides twice for 10 min each in 500 μl Wash Buffer B.
    3. Gently wash the slides for 1 min in 500 μl 0.01x Wash Buffer B.
    4. Let the slides dry at room temperature in the dark.
    5. Mount the slide glasses with cover glasses using a minimal volume of Duolink® In situ Mounting Medium with DAPI.
    6. Seal the edges with nail polish and incubate for 15 min at room temperature in the dark.

  9. Imaging with a microscope
    1. Analyze the images using fluorescence microscopy.
    2. After imaging, store the slides at -20 °C in the dark.

Data analysis

  1. PLA signals are detected with a Keyence BZ-8100 fluorescence microscope (Figure 3). 
  2. PLA signals are visible as red spots, and are detected in NSCLC cells in a manner dependent on the addition of both PLUS and MINUS probes, indicating the presence of EGFR homodimers. 
  3. Nuclei are stained blue with 4’,6-diamidino-2-phenylindole (DAPI). 
  4. The number of PLA signals per cell is quantified by counting the number of red spots in 20 cells using BZ Analyzer software.


    Figure 3. Detection of EGFR homodimerizations in NSCLC cell lines and resected specimens by PLA. Representative images of EGFR homodimers in H1975 cells (A) and in lung adenocarcinoma surgical specimens (B). The positive and negative controls are provided in Ota et al. (2017). Tumor cells surrounded by pink lines to count the number of red spots per cell by analytical software (B). Scale bar = 20 μm.

Notes

  1. The concentration of the primary antibodies before adding the conjugation buffer is desirably more than 1 mg/ml, and should at least be higher than that used for immunohistochemistry.
  2. Antibodies derived from any animal are permitted, we use rabbit antibodies.
  3. All experiments with detection reagents (from Procedure G onwards) are performed in a dark room.

Recipes

  1. Culture media (10% FBS)
    1. Open liquid DMEM or RPMI bottle (500 ml)
    2. Take out 55 ml with a sterile serological pipette and discard the liquid
    3. Add 50 ml FBS with a sterile serological pipette
    4. Add 5 ml Penicillin/Streptomycin solution
    5. Store at 4 °C
  2. Wash buffer A
    Dissolve the contents of one pouch in pure water to a final volume of 1,000 ml and store at 4 °C
  3. Wash buffer B
    Dissolve the contents of one pouch in pure water to a final volume of 1,000 ml and store at 4 °C

Acknowledgments

We thank Akiko Sato for technical assistance. We thank Sarah Williams, PhD, from Edanz Group (www.edanzediting.com) for editing a draft of this manuscript. This work was supported in part by JSPS KAKENHI Grant Number JP18K15927.

Competing interests

No potential conflicts of interest were disclosed.

References

  1. Duolink® In situ - Fluorescence User Guide. Sigma-Aldrich. (Accessed 4-July, 2018, at https://reedd.people.uic.edu/ReedLabPLA.pdf.)
  2. Duolink® In situ - Probemaker User Guide. Sigma-Aldrich. (Accessed 4-July, 2018, at https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Instructions/1/duolink-probemaker-user-manual.pdf.)
  3. Nagai, Y., Miyazawa, H., Huqun, Tanaka, T., Udagawa, K., Kato, M., Fukuyama, S., Yokote, A., Kobayashi, K., Kanazawa, M. and Hagiwara, K. (2005). Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res 65(16): 7276-7282.
  4. Ota, K., Harada, T., Otsubo, K., Fujii, A., Tsuchiya, Y., Tanaka, K., Okamoto, I. and Nakanishi, Y. (2017). Visualization and quantitation of epidermal growth factor receptor homodimerization and activation with a proximity ligation assay. Oncotarget 8(42): 72127-72132.

简介

致癌驱动因素在肿瘤发生以及肿瘤细胞存活和增殖中起重要作用。导致受体酪氨酸激酶组成性激活的表皮生长因子受体基因( EGFR )的突变已被鉴定为非小细胞肺癌(NSCLC)子集中的致癌驱动因子。通常采用基于PCR的检测来检测 EGFR 突变,但是没有在临床环境中建立检测不基于突变鉴定的EGFR活化的方法。我们描述了用于在NSCLC细胞系和组织样本中可视化和定量EGFR同源二聚化的邻近连接测定(PLA)。使用Probemaker将针对EGFR的兔单克隆抗体与PLUS或MINUS PLA寡核苷酸臂缀合。当两个EGFR单体非常接近时,发生PLUS和MINUS PLA探针的退火,并且退火的寡核苷酸复合物中的重复序列被扩增,然后被荧光标记的寡核苷酸探针识别。检测PLA信号并用荧光显微镜计数。我们证明了通过PLA分析在NSCLC细胞系和通过经支气管肺活检获得的组织样品中以定量方式检测EGFR同型二聚体。 PLA方法是用于检测和定量蛋白质 - 蛋白质相互作用的新工具,例如同型二聚体,异二聚体和融合蛋白。

【背景】目前通过基于PCR的测定法检测表皮生长因子受体基因( EGFR )的激活突变(Nagai et al。,2005)。然而,尚未确定临床环境中活化的EGFR的视觉检测和定量。 原位邻近连接测定(PLA)是一种使用Duolink ® 原位试剂(参见参考文献1和2)通过以下方法创建探针的技术寡核苷酸与抗体结合。当两种不同类型的PLA探针(PLUS和MINUS)非常接近(40nm)时,发生退火,产生扩增的环状DNA。来自每个检测到的PLA探针对的信号可视化为单个斑点,并且可以用荧光显微镜计数每个细胞的PLA信号数量(图1)。

PLA方法可用于检测紧邻的任何蛋白质 - 蛋白质相互作用。我们现在使用来自相同物种的一抗来应用PLA以显现和定量EGFR同源二聚化。此外,我们在NSCLC细胞系中检测到EGFR-人表皮生长因子受体2异二聚体和棘皮动物微管相关蛋白样4-间变性淋巴瘤激酶融合蛋白的形成(Ota 等,,2017)。该方法适用于其他二聚化和融合的检测。


图1. 原位 EGFR同源二聚体的PLA原理。 A.通过缀合PLA寡核苷酸和EGFR单克隆抗体产生的PLA探针与EGFR结合。 B.连接的寡核苷酸杂交并产生多个环状DNA分子。 C.荧光标记的与DNA环杂交的寡核苷酸的检测。 (图片来自Duolink ® In Situ 用户指南[参考文献1和2])。

关键字:致癌驱动, 表皮生长因子, 间变性淋巴瘤激酶, 非小细胞肺癌, 邻位连接检测, 二聚化

材料和试剂

  1. 标准移液器吸头,容量为10μl,20μl,100μl,200μl和1,000μl(Thermo Fisher Scientific,目录号:2140,2149P,ART 10REACH,ART 20P)
  2. Kimwipe废纸S-200(Crecia,目录号:62011)
  3. 12毫米直径无涂层盖玻片(Matsunami Glass,目录号:C012001)
  4. 24孔板(Greiner Bio-One,细胞培养多孔板,目录号:66)
  5. 染色罐(Matsunami,目录号:13号BT500)
  6. Dako Pen(Daido Sangyo,PAP-SPAP Pen Super-Liquid Blocker)
  7. 血清移液器
  8. 滑动眼镜
  9. NSCLC细胞系:H1975细胞
  10. Roswell Park Memorial Institute(RPMI)1640 medium(Invitrogen,Gibco,目录号:61870-036)
  11. Dulbecco的改良Eagle's培养基(DMEM)(Invitrogen,Gibco,目录号:12634010)
  12. 胎牛血清(FBS)(西格玛,目录号:14C438FBS)
  13. 青霉素/链霉素溶液(Invitrogen,目录号:15140122)
  14. 磷酸盐缓冲盐水(PBS)(LSI,目录号:RM102-PN)
  15. PBS中4%多聚甲醛(Wako,目录号:16320145)
  16. ImmunoSaver(Wako,目录号:9706192)
  17. Duolink ® 原位寡核苷酸PLUS(-20°C)(Sigma-Aldrich,目录号:DUO92009)
  18. Duolink ® 原位寡核苷酸MINUS(-20°C)(Sigma-Aldrich,目录号:DUO92010)
  19. 共轭缓冲液(-20°C)(西格玛奥德里奇,目录号:DUO92009)
  20. 停止试剂(-20°C)(西格玛奥德里奇,目录号:DUO92009)
  21. 储存溶液(-20°C)(Sigma-Aldrich,目录号:DUO92009)
  22. 20x分析试剂(-20°C)(Sigma-Aldrich,目录号:DUO92009)
  23. 封闭溶液(4°C)(Sigma-Aldrich,目录号:DUO92009)
  24. PLA探针稀释剂(4°C)(Sigma-Aldrich,目录号:DUO92009)
  25. 兔EGFR单克隆抗体(Abcam,目录号:ab52894)
  26. 5x连接缓冲液(-20°C)(Sigma-Aldrich,目录号:DUO92008)
  27. 1x Ligase(-20°C)(Sigma-Aldrich,目录号:DUO92008)
  28. 5x扩增红(-20°C)(Sigma-Aldrich,目录号:DUO92008)
  29. 1x聚合酶(-20°C)(Sigma-Aldrich,目录号:DUO92008)
  30. 带有DAPI的Duolink ® 原位封固剂(Sigma-Aldrich,目录号:DUO82040)
  31. 10%中性缓冲福尔马林
  32. 石蜡
  33. 二甲苯
  34. 乙醇溶液
  35. 指甲油
  36. 文化媒体(10%FBS)(见食谱)
  37. 洗涤缓冲液A(Sigma-Aldrich,目录号:DUO82049,参见食谱)
  38. 洗涤缓冲液B(Sigma-Aldrich,目录号:DUO82049,参见食谱)

设备

  1. 手动移液器:10μl,20μl,100μl,200μl和1,000μl(Mettler Toledo,目录号:Pipet-Lite XLS + 17014409,17014412,17014408,17014411,17014407)
  2. 镊子
  3. 冰箱
  4. Direct-Q ® 5 UV远程净水系统(Merck,型号:Direct-Q ® UV 5 Remote,目录号:ZRQSVR5WW)
  5. 高压灭菌器(Tomy,目录号:LSX-500)
  6. 涡旋混合器(科学工业,型号:Vortex-Genie 2,目录号:S1-0286)
  7. 湿度室(孵化室,Cosmo Bio,目录号:10DO)
  8. 酶冻结块(-20°C)(Eppendorf,目录号:3880001018)
  9. 荧光显微镜(Keyence,型号:BZ-8100)

软件

  1. BZ分析仪软件(Keyence)
  2. Excel(微软)
  3. GraphPad Prism 5.0(GraphPad软件)

程序

注意:所有程序都是针对四个样本执行的。

  1. 细胞的制备(图2)
    1. 将12毫米直径的盖玻片放入24孔板的每个孔中。
    2. 在盖玻璃上,在500μl推荐的培养基中,在37℃,5%CO 2 下使细胞生长至60%汇合。
    3. 从孔中取出培养基,每次用1 ml PBS洗涤两次。 
    4. 在室温下加入500μl4%多聚甲醛15分钟。
    5. 从孔中取出溶液,用1ml PBS洗涤两次。
    6. 用镊子从井中取出盖玻片。


    图2.使用细胞系制备PLA方法。 A.在平板的每个孔中盖上眼镜(左)。细胞在培养基中的盖玻片上生长(右)。 B.使用DAPI安装盖玻片并用指甲油密封的后PLA载玻片。

  2. 制备肿瘤标本
    1. 将手术标本固定在10%中性缓冲福尔马林中,将其嵌入石蜡中,切片厚度为4μm。
    2. 用二甲苯除去石蜡,用分级系列的乙醇溶液再水化,并用PBS洗涤两次。
    3. 将300μlImmunoSaver添加到染色罐中的60ml Milli-Q水中。
    4. 将载玻片在98℃下在染色罐中高压灭菌45分钟并在室温下孵育。
    5. 用PBS洗涤载玻片两次。
    6. 用Dako Pen在约1 cm 2 的肿瘤标本周围画一个圆圈。

  3. PLA寡核苷酸与抗体的缀合
    1. 在室温下解冻缀合缓冲液并涡旋。
    2. 将2.1μl缀合缓冲液加入21μl兔单克隆抗EGFR抗体中,并用移液管轻轻混合。
    3. 将来自步骤C2的抗体溶液转移至一小瓶寡核苷酸PLUS或寡核苷酸MINUS,并用移液管轻轻混合。
    4. 在室温下孵育过夜。
    5. 涡旋停止试剂溶液并将2.1μl加入到步骤C3的溶液中并在室温下孵育30分钟。
    6. 涡旋储存溶液,向步骤C5中制备的溶液中加入25.2μl,并在4℃下储存。
    7. 存储的解决方案可在一个月内使用。

  4. 封锁
    1. 涡旋阻断溶液并将40μl转移至每个样品。
    2. 将载玻片放入预热的湿度室中,在潮湿的37℃,5%CO 2 培养箱中孵育30分钟。

  5. 解放军探测器
    1. 稀释PLA探针稀释液中步骤C6的缀合抗体。
      1. 通过混合10μl20x储备溶液和190μlPLA探针稀释剂制备200μl1x工作分析试剂。
      2. 将8μlPLUS或MINUS抗体溶液(步骤C6)加入72μl工作测定试剂(来自步骤E1a)并在室温下孵育20分钟。
    2. 使用Kimwipe废纸板轻轻地从样品中取出封闭溶液,不要接触细胞。
    3. 在总稀释PLA探针PLUS溶液和MINUS溶液中加入38μl(19μlPLUS探针和19μlMINUS探针)至每个样品。
    4. 用移液管轻轻混合。
    5. 在室温下在湿度室中孵育2小时。

  6. 结扎
    1. 在124μlMilli-Q水中稀释32μl连接缓冲液(5x)并混合。
    2. 使用Kimwipe废纸片轻轻地从样品中取出PLA探针溶液,不要接触细胞。
    3. 在500μl洗涤缓冲液A中轻轻洗涤载玻片两次,每次5分钟。
    4. 将4μl连接酶添加至156μl连接缓冲液(1x)并涡旋。
    5. 每个样品中加入39μl溶液。
    6. 将载玻片放入预热的湿度室中,在潮湿的37℃,5%CO 2 培养箱中孵育30分钟。

  7. 放大
    1. 在126μlMilli-Q水中稀释32μl扩增红色(5x)并混合。
    2. 使用Kimwipe废纸片轻轻地从样品中取出连接溶液,不要接触细胞。
    3. 在500μl洗涤缓冲液A中轻轻洗涤载玻片两次,每次2分钟。
    4. 将2μl聚合酶溶液加入158μl扩增红(1x)并涡旋。
    5. 每个样品中加入39μl溶液。
    6. 将载玻片放入预热的湿度室中,并用加湿的37℃,5%CO 2 培养箱孵育30分钟。

  8. 准备成像
    1. 使用Kimwipe废纸片轻轻地从样品中取出扩增聚合酶溶液,不要接触细胞。
    2. 在500μl洗涤缓冲液B中轻轻洗涤载玻片两次,每次10分钟。
    3. 在500μl0.01x洗涤缓冲液B中轻轻洗涤载玻片1分钟。
    4. 将载玻片在室温下在黑暗中干燥。
    5. 使用最小体积的Duolink ® 原位安装介质和DAPI,用盖玻片安装载玻片。
    6. 用指甲油密封边缘,在室温下避光孵育15分钟。

  9. 用显微镜成像
    1. 使用荧光显微镜分析图像。
    2. 成像后,将载玻片保存在-20°C的黑暗中。

数据分析

  1. 用Keyence BZ-8100荧光显微镜检测PLA信号(图3)。 
  2. PLA信号作为红色斑点可见,并且在NSCLC细胞中以依赖于PLUS和MINUS探针的添加的方式检测,表明存在EGFR同型二聚体。 
  3. 细胞核用4',6-二脒基-2-苯基吲哚(DAPI)染成蓝色。 
  4. 通过使用BZ Analyzer软件计算20个细胞中的红点数来量化每个细胞的PLA信号数量。


    图3.通过PLA检测NSCLC细胞系和切除标本中的EGFR同源二聚化。 H1975细胞(A)和肺腺癌手术标本(B)中EGFR同型二聚体的代表性图像。 Ota 等人提供了阳性和阴性对照(2017)。用粉红色线包围的肿瘤细胞通过分析软件计算每个细胞的红色斑点数(B)。比例尺=20μm。

笔记

  1. 在添加缀合缓冲液之前,一抗的浓度理想地大于1mg / ml,并且应该至少高于用于免疫组织化学的浓度。
  2. 允许来自任何动物的抗体,我们使用兔抗体。
  3. 所有使用检测试剂的试验(从程序G开始)都在暗室中进行。

食谱

  1. 文化传媒(10%FBS)
    1. 开放式液体DMEM或RPMI瓶(500 ml)
    2. 用无菌血清移液管取出55毫升,弃去液体
    3. 用无菌血清移液管加入50ml FBS
    4. 加入5毫升青霉素/链霉素溶液
    5. 储存在4°C
  2. 洗涤缓冲液A
    将一个小袋中的内容物溶解在纯水中至最终体积为1,000ml并储存在4℃
  3. 洗涤缓冲液B
    将一个小袋中的内容物溶解在纯水中至最终体积为1,000毫升,并储存在4°C

致谢

我们感谢Akiko Sato的技术支持。我们感谢Edanz Group的Sarah Williams博士( www.edanzediting.com )编辑本稿件的草稿。这项工作部分得到了JSPS KAKENHI Grant No. JP18K15927的支持。

利益争夺

没有披露潜在的利益冲突。

参考

  1. Duolink ® 原位 - 荧光用户指南。 Sigma-Aldrich公司。 (2018年7月4日访问, https://reedd.people.uic.edu/ReedLabPLA。 PDF格式。)
  2. Duolink ® In Situ - Probemaker用户指南。 Sigma-Aldrich公司。 (于2018年7月4日访问, https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Instructions/1/duolink-probemaker-user-manual.pdf。 )
  3. Nagai,Y.,Miyazawa,H.,Huqun,Tanaka,T.,Udagawa,K.,Kato,M.,Fukuyama,S.,Yokote,A.,Kobayashi,K.,Kanazawa,M。和Hagiwara,K 。(2005)。 快速揭示的非小细胞肺癌细胞系中表皮生长因子受体的遗传异质性和灵敏检测系统,肽核酸锁定核酸PCR钳。 Cancer Res 65(16):7276-7282。
  4. Ota,K.,Harada,T.,Otsubo,K.,Fujii,A.,Tsuchiya,Y.,Tanaka,K.,Okamoto,I。和Nakanishi,Y。(2017)。 使用邻近连接试验可视化和定量表皮生长因子受体同源二聚化和活化。 Oncotarget 8(42):72127-72132。
登录/注册账号可免费阅读全文
  • 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: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ota, K. and Harada, T. (2018). Protocol for in situ Proximity Ligation Assay (PLA) and Microscopy Analysis of Epidermal Growth Factor Receptor (EGFR) Homodimerization. Bio-protocol 8(21): e3067. DOI: 10.21769/BioProtoc.3067.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。