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DNA in situ Hybridizations for VEGFA Gene Locus (6p12) in Human Tumor Tissue
人肿瘤组织中VEGFA基因座(6p12)的DNA原位杂交

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Abstract

Over the last decades numerous regulators of angiogenesis have been identified and characterized. Among the others the vascular endothelial growth factor (VEGFA) appears undoubtedly important in several pathophysiological processes. Moreover, VEGFA represents one of the most attractive targets of anticancer therapy, given its major role in the growth and development of different tumor types. Here we describe a method to detect the copy number variation (CNV) status of the VEGFA gene by fluorescence in situ hybridization (FISH). FISH analysis is a reliable method for investigating VEGFA amplification or increased gene copy number and may represent an alternative method to immunohistochemical analysis for investigating the deregulation of VEGFA expression levels.

Keywords: VEGFA(VEGFA), TMA(TMA), FISH(鱼), 6p12(6p12), CNV(CNV)

Materials and Reagents

  1. BAC clone No. RPCIB753M0921Q (GenomeCUBE)
  2. LB medium (Sigma-Aldrich, catalog number: L3522-250G )
  3. Large-construct kit (QIAGEN, catalog number: 12462 )
  4. SP6 (EuroFins MGW Operon LLC A Eurofins Genomics Company, catalog number: SP050-1 ) and T7 (EuroFins MGW Operon LLC A Eurofins Genomics Company, catalog number: SP401 ) primers
  5. AluI restriction enzyme (Life Technologies, InvitrogenTM, catalog number: 45200-029 )
  6. Cy3-dUTP (GE Healthcare Dharmacon, catalog number: 45-000-738 )
  7. BioPrime array CGH kit (module) (Life Technologies, InvitrogenTM, catalog number: 18095-012 ) (it includes 2.5 Random primers solution, 10x dUTP nucleotide mix, Exo-Klenow fragment)
  8. Amicon Ultra-0.5 centrifugal filter unit with Ultracel-30 membrane (Merck KGaA, catalog number: UFC503096 )
  9. Human Cot-1 DNA (Life Technologies, InvitrogenTM, catalog number: 15279-011 )
  10. DAPI solution (1 µg/ml) (Abbott Laboratories, catalog number: 06J49-001 )
  11. Spectrum Green-labeled Chr6 centromeric probe (CEP6) and hybridization buffer (Abbott Laboratories, catalog number: 06J37-016 and 07J36-001 )
  12. Micron-centrifugal filters (Merck KGaA, catalog number: MRCF0R030 )
  13. TE Buffer, 1x Solution 500 ml (USB, catalog number: 75893 )
  14. 20x SSC (Abbott Laboratories, catalog number: 02J10-032 )
  15. 0.3 % NP40 (Abbott Laboratories, catalog number: 07J05-001 )
  16. Pretreatment reagent VP 2000 (Abbott Laboratories, catalog number: 02J06-030 )
  17. Protease 1 (250 mg) (Abbott Laboratories, catalog number: 02J08-032 )
  18. Ethanol (Sigma- Aldrich, catalog number: 0 2860 )
  19. Rubber cement (Carfa AG, catalog number: 00494 )
  20. Protease 1 (250 mg in 500 ml protease buffer) (Sauter et al., 1995)
  21. Wash buffer (0.4 SSC + 0.3% NP40-pH 7-7.5) (Sauter et al., 1995)

Equipment

  1. Incubator for slides (Memmert GmbH + Co.KG, Mode: ll IPP 300 )
  2. Pipette (Eppendorf)
  3. Nanodrop spectrophotometer assay (Thermo Fisher Scientific, model: NanoDrop 2000 )
  4. Slide (Thermo Fisher Scientific, catalog number: No. 10143352 )
  5. Cover slide (Biosystem 24 x 50 mm, catalog number: 14071135638 and R. Langenbrinck 21 x 26 mm, catalog number: 01-2126 )
  6. Hotplate for denaturation (Medax GmbH & Co.KG, catalog number: 12895 )
  7. Zeiss fluorescence microscope using a 63x objective (Carl Zeiss AG, model: Axioskop 40 )
  8. AxioVision software (Carl Zeiss AG, model: SE64 Rel.4.9 )

Procedure

  1. How to make the probe
    1. Inoculate starter culture with the BAC clone (e.g. glycerol stock) in 2-5 ml LB medium, containing the appropriate selective antibiotic (chloramphenicol).
    2. Incubate over-night 25/30 °C, shaking at 200 rpm.
    3. Dilute 0.5 ml of the starter culture in 500 ml selective LB medium (1/500 to 1/1,000), containing the appropriate antibiotic (chloramphenicol).
    4. Incubate over-night with shaking (200 rpm).
    5. Isolate the BAC-DNA using the Large-Construct Kit according to the manufacturer’s instructions.
    6. Verify BAC identity by sequencing using 1 μg of isolated DNA and 20 pmol of SP6, T7 primers, respectively.
    7. Digest ~1 μg BAC-DNA with AluI restriction enzyme according to the manufacturer’s instructions.
    8. Suspend the digested BAC-DNA in sterile water to a final volume of 21 μl (BioPrime array CGH kit).
    9. Add 20 μl of 2.5x Random primers solution (BioPrime array CGH kit) to each sample and incubate at 95 °C for 5 min.
    10. On ice, add 5 μl of 10x dUTP Nucleotide Mix (BioPrime array CGH kit).
    11. Add 3 μl of Cy3-dUTP.
    12. Add 1 μl of Exo-Klenow fragment (BioPrime array CGH kit).
    13. Mix gently and perform a quick spin down (5 sec) and incubate at 37 °C for 2 h.
    14. Add 5 μl of Stop Buffer to each tube and place on ice (BioPrime array CGH kit).

      Labeling mix  

      Reagent Added per Sample   
      Volume Added per Sample
      2.5x Random primers solution (BioPrime array CGH kit) and incubate at 95 °C for 5 min   
      20 µl
      On ice, 10x dUTP Nucleotide Mix   
      5 µl
      Cy3-dUTP   
      3 µl
      Exo-Klenow fragment   
      1 µl
      Mix gently and perform a quick spin down (5 sec) then incubate at 37 °C for 2 h   

      Stop Buffer to each tube and place on ice (long term storage at -20 °C)   
      5 µl

    15. Label the Micron tubes on both the lid and the side with sample identifiers, as the lids tend to break. Also label the side of the filter with an identifier.
    16. Load the Micron filter into the 1.5 ml tubes provided with the kit, so that the filter is sifting on the ridges inside the tube.
    17. Pipette 450 μl of TE 1x into each filter.
    18. Load the samples into the filters.
    19. Centrifuge at 8,000 x g for 10 min at room temperature.
    20. Check the filter (visual inspection by eyes) to make sure the Cy3-Dye labelled DNA has stuck to it.
    21. Add 500 μl of TE to the filter and centrifuge at 8,000 x g for 10 min.
    22. Discard the flow through and place in a new 1.5 ml filter tube upside-down to elute.
    23. Centrifuge at 8,000 x g for 1 min.
    24. Check the filter to make sure the entire sample is eluted from the filter (store samples short-term at 4 °C. Store long-term at -20 °C).
    25. Add 20 μl of Human Cot-1 DNA.
    26. Add 5 μl of CEP 6.
    27. Add 35 μl of hybridization buffer (keep in ice or store at -20 °C).

  2. How to prepare the tissue
    1. Pretreat FFPE tissue microarrays or FFPE whole tissue sections (7 μm thick) 10 min in the Xylene-1 min in 100% EtOH-1 min in 90% EtOH-1 min in 70% EtOH-1 min in H2O.
    2. Continue pretreating the tissue sections for 15 min in the pre-warmed paraffin pretreatment reagent VP2000 (80 °C).
    3. Immerse the tissue sections for 1 min in the H2O.
    4. Immerse the tissue sections for 2 h into the pre-warmed 37 °C protease buffer (Protease 1 - 250 mg in 500 ml Protease Buffer).
    5. Immerse the tissue sections for 1 min in the H2O.
    6.  Immerse the tissue sections for 1 min in the 70% EtOH-1 min in the 90% EtoH-1 min in the 100% EtOH-1 min in the H2O.
    7. Let the tissue section air-dry at room temperature for approximately 10 min.

  3. Hybridization
    1. Apply 10-20 μl of the probe on the tissue sections and cover it.
    2. Apply the rubber cement to prevent the probe from leaking.
    3. Leave the tissue sections for 20 min at 75 °C.
    4. Incubate the tissue sections over-night at 37 °C.
    5. Uncover the tissue sections.
    6. Wash the tissue sections in the wash buffer at room temperature 5 min.
    7. Wash the tissue sections in the wash buffer, pre-warmed at 48 °C between 2-5 min (when you have background wash longer and with higher temperature).
    8. Immerse the tissue sections 1 min in the H2O.
    9. Let the tissue sections dry protecting it from the light.
    10. Add 10-20 μl of DAPI solution (1,000 ng/ml) to the tissue sections.
    11. Cover the tissue sections with cover glass 24 x 50 mm.
    12. Place the tissue sections 15 min at 4 °C.
    13. Evaluate the tissue sections using a fluorescence microscope (63x objective).
    14. Count a minimum of 100 tumor nuclei signals in four separate regions of the tissue section independently.

Representative data

FISH results (representative pictures are shown in Figure 1) were interpreted according to: (1) absolute Chr6 copy number and (2) the ratio VEGFA gene/Chr6 copy number. We classified as not amplified samples with a VEGFA/Chr6 ratio of <1.8 and an average VEGFA copy number <4.0 signals per cell; equivocal with a VEGFA/Chr6 ratio <2.0 and an average VEGFA copy number ≥ 4.0 and <6.0 signals per cell and amplified with a VEGFA/Chr6 ratio of <2.2 and an average VEGFA copy number of ≥6.0 signals per cell or a VEGFA/Chr6 ratio ≥2.2 with an average VEGFA copy number of ≥4.0 signals per cell, as proposed by the ASCO/CAP guidelines for HER2 amplification in breast cancer (Wolff et al., 2013). Polysomy of Chr6 was defined as an average of the Chr6 copy number. When the average was included between 2.26 and 3.75, the polysomy 6 was defined as low whereas, when the average was >3.75 the polysomy 6 was defined high (Andreozzi et al., 2014; Sauter et al., 1995; Wolff et al., 2007; Salido et al., 2005; Torrisi et al., 2007; Tapia et al., 2007; Ma et al., 2005).


Figure 1. Fluorescence in situ hybridization (FITC+Rhodamine+DAPI) of the VEGFA gene locus 6p12 in colorectal cancer (CRC), (A) non-amplified CRC sample, (B) amplified CRC sample. Red dots highlight VEGFA gene, green dots highlight Chr6 centromere.

Acknowledgments

We would like to acknowledge Matthias Matter, MD, PhD, Cristina Quintavalle, PhD and Christian Ruiz, PhD, for their suggestions and critical comments during the establishment phase of this methodology. This protocol represents a modified enhanced type of the previously used version (Sauter et al., 1995; Vlajnic et al., 2011; Andreozzi et al., 2014).

References

  1. Andreozzi, M., Quagliata, L., Gsponer, J. R., Ruiz, C., Vuaroqueaux, V., Eppenberger-Castori, S., Tornillo, L. and Terracciano, L. M. (2014). VEGFA gene locus analysis across 80 human tumour types reveals gene amplification in several neoplastic entities. Angiogenesis 17(3): 519-527.
  2. Ma, Y., Lespagnard, L., Durbecq, V., Paesmans, M., Desmedt, C., Gomez-Galdon, M., Veys, I., Cardoso, F., Sotiriou, C., Di Leo, A., Piccart, M. J. and Larsimont, D. (2005). Polysomy 17 in HER-2/neu status elaboration in breast cancer: effect on daily practice. Clin Cancer Res 11(12): 4393-4399.
  3. Salido, M., Tusquets, I., Corominas, J. M., Suarez, M., Espinet, B., Corzo, C., Bellet, M., Fabregat, X., Serrano, S. and Sole, F. (2005). Polysomy of chromosome 17 in breast cancer tumors showing an overexpression of ERBB2: a study of 175 cases using fluorescence in situ hybridization and immunohistochemistry. Breast Cancer Res 7(2): R267-273.
  4. Sauter, G., Moch, H., Carroll, P., Kerschmann, R., Mihatsch, M. J. and Waldman, F. M. (1995). Chromosome-9 loss detected by fluorescence in situ hybridization in bladder cancer. Int J Cancer 64(2): 99-103.
  5. Tapia, C., Savic, S., Wagner, U., Schonegg, R., Novotny, H., Grilli, B., Herzog, M., Barascud, A. D., Zlobec, I., Cathomas, G., Terracciano, L., Feichter, G. and Bubendorf, L. (2007). HER2 gene status in primary breast cancers and matched distant metastases. Breast Cancer Res 9(3): R31.
  6. Torrisi, R., Rotmensz, N., Bagnardi, V., Viale, G., Curto, B. D., Dell'orto, P., Veronesi, P., Luini, A., D'Alessandro, C., Cardillo, A., Goldhirsch, A. and Colleoni, M. (2007). HER2 status in early breast cancer: relevance of cell staining patterns, gene amplification and polysomy 17. Eur J Cancer 43(16): 2339-2344.
  7. Vlajnic, T., Andreozzi, M. C., Schneider, S., Tornillo, L., Karamitopoulou, E., Lugli, A., Ruiz, C., Zlobec, I. and Terracciano, L. (2011). VEGFA gene locus (6p12) amplification identifies a small but highly aggressive subgroup of colorectal cancer [corrected] patients. Mod Pathol 24(10): 1404-1412.
  8. Wolff, A. C., Hammond, M. E., Hicks, D. G., Dowsett, M., McShane, L. M., Allison, K. H., Allred, D. C., Bartlett, J. M., Bilous, M., Fitzgibbons, P., Hanna, W., Jenkins, R. B., Mangu, P. B., Paik, S., Perez, E. A., Press, M. F., Spears, P. A., Vance, G. H., Viale, G., Hayes, D. F., American Society of Clinical, O. and College of American, P. (2013). Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol 31(31): 3997-4013.
  9. Wolff, A. C., Hammond, M. E., Schwartz, J. N., Hagerty, K. L., Allred, D. C., Cote, R. J., Dowsett, M., Fitzgibbons, P. L., Hanna, W. M., Langer, A., McShane, L. M., Paik, S., Pegram, M. D., Perez, E. A., Press, M. F., Rhodes, A., Sturgeon, C., Taube, S. E., Tubbs, R., Vance, G. H., van de Vijver, M., Wheeler, T. M., Hayes, D. F., American Society of Clinical, O. and College of American, P. (2007). American society of clinical oncology/college of american pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25(1): 118-145.

简介

在过去的几十年中,已经鉴定和表征了许多血管生成的调节剂。 其中血管内皮生长因子(VEGFA)在几种病理生理过程中无疑是重要的。 此外,VEGFA代表抗癌治疗的最有吸引力的靶标之一,因为其在不同肿瘤类型的生长和发展中的主要作用。 在这里我们描述了通过荧光原位杂交(FISH)检测VEGFA基因的拷贝数变异(CNV)状态的方法。 FISH分析是用于研究VEGFA扩增或增加的基因拷贝数的可靠方法,并且可以代表用于研究VEGFA表达水平的失调的免疫组织化学分析的替代方法。

关键字:VEGFA, TMA, 鱼, 6p12, CNV

材料和试剂

  1. BAC克隆No.RPCIB753M0921Q(GenomeCUBE)
  2. LB培养基(Sigma-Aldrich,目录号:L3522-250G)
  3. 大构建试剂盒(QIAGEN,目录号:12462)
  4. SP6(EuroFins MGW Operon LLC A Eurofins Genomics Company,目录号:SP050-1)和T7(EuroFins MGW Operon LLC A Eurofins Genomics Company,目录号:SP401)引物
  5. (Life Technologies,Invitrogen TM ,目录号:45200-029)。
  6. Cy3-dUTP(GE Healthcare Dharmacon,目录号:45-000-738)
  7. BioPrime阵列CGH试剂盒(模块)(Life Technologies,Invitrogen ,目录号:18095-012)(其包括2.5个随机引物溶液,10x dUTP核苷酸混合物,Exo-Klenow片段)
  8. 带有Ultracel-30膜的Amicon Ultra-0.5离心过滤器单元(Merck KGaA,目录号:UFC503096)
  9. 人Cot-1 DNA(Life Technologies,Invitrogen TM ,目录号:15279-011)
  10. DAPI溶液(1μg/ml)(Abbott Laboratories,目录号:06J49-001)
  11. 光谱绿色标记的Chr6着丝粒探针(CEP6)和杂交缓冲液(Abbott Laboratories,目录号:06J37-016和07J36-001)
  12. 微米离心过滤器(Merck KGaA,目录号:MRCF0R030)
  13. TE缓冲液,1x Solution 500ml(USB,目录号:75893)
  14. 20x SSC(Abbott Laboratories,目录号:02J10-032)
  15. 0.3%NP40(Abbott Laboratories,目录号:07J05-001)
  16. 预处理试剂VP 2000(Abbott Laboratories,目录号:02J06-030)
  17. 蛋白酶1(250mg)(Abbott Laboratories,目录号:02J08-032)
  18. 乙醇(Sigma-Aldrich,目录号:02860)
  19. 橡胶胶泥(Carfa AG,目录号:00494)
  20. 蛋白酶1(250mg在500ml蛋白酶缓冲液中)(Sauter等人,1995)
  21. 洗涤缓冲液(0.4SSC + 0.3%NP40-pH7-7.5)(Sauter等人,1995)

设备

  1. 载玻片保温箱(Memmert GmbH + Co.KG,模式:IPP 300)
  2. 移液管(Eppendorf)
  3. Nanodrop分光光度计测定(Thermo Fisher Scientific,型号:NanoDrop 2000)
  4. Slide(Thermo Fisher Scientific,目录号:10143352)
  5. 盖玻片(Biosystem 24×50mm,目录号:14071135638和R. Langenbrinck 21×26mm,目录号:01-2126)
  6. 用于变性的热板(Medax GmbH& Co.KG,目录号:12895)
  7. Zeiss荧光显微镜,使用63x物镜(Carl Zeiss AG,型号:Axioskop 40)
  8. AxioVision软件(Carl Zeiss AG,型号:SE64 Rel.4.9)

程序

  1. 如何使探头
    1. 用BAC克隆(例如甘油原液)接种起子培养物 2-5 ml LB培养基,含有合适的选择性抗生素 (氯霉素)。
    2. 孵育过夜25/30°C,以200 rpm摇动
    3. 在500ml选择性LB培养基中稀释0.5ml起始培养物 (1/500至1/1,000),含有合适的抗生素 (氯霉素)。
    4. 孵育过夜,摇动(200 rpm)。
    5. 根据制造商的说明,使用大构建试剂盒分离BAC-DNA
    6. 通过使用1μg分离的DNA和20pmol SP6,T7引物分别测序来验证BAC身份
    7. 根据制造商的说明书用IluI限制酶消化〜1μgBAC-DNA。
    8. 将消化的BAC-DNA在无菌水中悬浮至终体积为21μl(BioPrime array CGH试剂盒)。
    9. 向每个样品中加入20μl的2.5x随机引物溶液(BioPrime array CGH试剂盒),并在95℃下孵育5分钟。
    10. 在冰上,加入5μl10x dUTP核苷酸混合物(BioPrime array CGH试剂盒)
    11. 加入3μlCy3-dUTP
    12. 加入1μl的Exo-Klenow片段(BioPrime array CGH试剂盒)
    13. 轻轻混匀,快速离心(5秒),37°C孵育2小时
    14. 每管加入5μl终止缓冲液并置于冰上(BioPrime array CGH试剂盒)
      标签组合  

      试剂每个样品添加   
      每个样品添加的量
      2.5x随机引物溶液(BioPrime array CGH试剂盒),并在95℃下孵育5分钟 
      20微升
      在冰上,10x dUTP核苷酸混合物   
      5微升
      Cy3-dUTP   
      3微升
      Exo-Klenow片段   
      1微升
      轻轻混合,快速离心(5秒),然后在37℃孵育2小时。

      停止缓冲液到每个管,并放置在冰上(长期储存在-20℃)   
      5微升

    15. 用样品在盖子和侧面标记Micron管 标识符,因为盖子倾向于破裂。 也标签的一面 过滤器标识符。
    16. 将Micron过滤器装入1.5 ml试管提供的试管,使过滤器在筛上 在管内的脊
    17. 吸取450微升TE 1x到每个过滤器
    18. 将样品装入过滤器。
    19. 在室温下以8000xg离心10分钟。
    20. 检查过滤器(目视检查),以确保Cy3-Dye标记的DNA粘附在其上
    21. 向过滤器中加入500μlTE,并以8,000×g离心10分钟。
    22. 弃去流过,并置于新的1.5 ml过滤管中倒置以洗脱
    23. 以8000xg离心1分钟。
    24. 检查过滤器,以确保整个样品从中洗脱 过滤器(在4°C短期储存样品,长期储存在-20°C)
    25. 加入20μlHuman Cot-1 DNA
    26. 加入5μlCEP 6.
    27. 加入35微升的杂交缓冲液(保存在冰上或-20°C存储)

  2. 如何准备组织
    1. 预处理FFPE组织微阵列或FFPE全组织切片(7μm 厚)在90%EtOH-1分钟的100%EtOH-1分钟的二甲苯-1分钟内10分钟 在70%EtOH-1分钟的H 2 O中
    2. 继续在预热的石蜡预处理试剂VP2000(80℃)中预处理组织切片15分钟。
    3. 将组织切片在H 2 O中浸泡1分钟
    4. 将组织切片浸入预热的37°C 2小时 蛋白酶缓冲液(蛋白酶1-250mg,在500ml蛋白酶缓冲液中)
    5. 将组织切片在H 2 O中浸泡1分钟
    6. 将组织切片在H 2 O中的100%EtOH-1分钟的90%EtoH-1分钟中浸泡在70%EtOH-1分钟中1分钟。
    7. 让组织切片在室温下空气干燥约10分钟。

  3. 杂交
    1. 在组织切片上涂抹10-20μl的探针并盖住
    2. 使用橡胶水泥防止探针泄漏。
    3. 将组织切片在75℃保持20分钟。
    4. 在37℃下孵育过夜的组织切片。
    5. 揭开组织切片。
    6. 在室温下洗涤洗涤缓冲液中的组织切片5分钟
    7. 洗涤组织切片在洗涤缓冲液,预热在48°C 在2-5分钟之间(当你有背景洗涤更长和更高 温度)。
    8. 在H 2 O中浸泡组织切片1分钟。
    9. 让组织切片干燥保护它不受光。
    10. 向组织切片中加入10-20μlDAPI溶液(1,000ng/ml)
    11. 用24 x 50 mm的玻璃罩覆盖组织切片。
    12. 将组织切片在4℃下放置15分钟。
    13. 使用荧光显微镜(63x物镜)评估组织切片。
    14. 在组织切片的四个独立区域独立计数最少100个肿瘤细胞核信号。

代表数据

FISH结果(代表性图片显示于图1中)根据以下进行解释:(1)绝对Chr6拷贝数和(2)比率VEGFA基因/Chr6拷贝数。我们分类为具有<1.8的VEGFA/Chr6比率和平均VEGFA拷贝数<4.0个信号/细胞的扩增样品;其中VEGFA/Chr6比率<2.0且每个细胞的平均VEGFA拷贝数≥4.0且<6.0个信号,并且以<2.2的VEGFA/Chr6比率和每个细胞的平均VEGFA拷贝数≥6.0个信号进行扩增,或VEGFA/Chr6比率≥2.2,每个细胞的平均VEGFA拷贝数≥4.0个信号,如ASCO/CAP指南对乳腺癌中HER2扩增所提出的(Wolff等人,2013)。 Chr6的多基因切割定义为Chr6拷贝数的平均值。当平均值包括在2.26和3.75之间时,多形性6被定义为低,而当平均值> 3.75时,多形性6被定义为高(Andreozzi等人,2014; Sauter al。,2005)。 tapia等人,2007;="" ,2007;="" al。,="" torrisi="" al。,2005;="" salido="" al。,2007;="" wolff="" al。,1995;="">

图1.结肠直肠癌(CRC)中的VEGFA基因座6p12的荧光原位杂交(FITC +罗丹明+ DAPI),(A)非扩增的CRC样品,(B)扩增的CRC样品。红点突出显示VEGFA基因,绿点突出显示Chr6着丝粒。

al。,2005)。 tapia等人,2007;="" ,2007;="" al。,="" torrisi="" al。,2005;="" salido="" al。,2007;="" wolff="" al。,1995;=""> 致谢

al。,2005)。 tapia等人,2007;="" ,2007;="" al。,="" torrisi="" al。,2005;="" salido="" al。,2007;="" wolff="" al。,1995;=""> 我们要感谢Matthias Matter,MD,PhD,Cristina Quintavalle,PhD和Christian Ruiz,PhD,他们在本方法建立阶段提出的建议和批评意见。该协议表示先前使用的版本的修改的增强类型(Sauter等人,1995; Vlajnic等人,2011; Andreozzi等人, em>,2014)。

al。,2005)。 tapia等人,2007;="" ,2007;="" al。,="" torrisi="" al。,2005;="" salido="" al。,2007;="" wolff="" al。,1995;=""> 参考文献

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引用:Andreozzi, M., Quagliata, L., Burmeister, K., Arabi, L., Schneider, S., Tornillo, L. and Terracciano, L. (2015). DNA in situ Hybridizations for VEGFA Gene Locus (6p12) in Human Tumor Tissue. Bio-protocol 5(15): e1553. DOI: 10.21769/BioProtoc.1553.
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