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ImmunoFISH for Adherent Cultured Mammalian Cells
附着培养哺乳动物细胞的ImmunoFISH试验   

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Abstract

This protocol is optimized for immunoFISH staining of adherent cultured mammalian cells. It combines immunofluorescence for DNA damage response factors (e.g. 53BP1) and FISH against telomeric DNA.

Keywords: ImmunoFISH(immunofish), DNA damage response(DNA损伤反应), Telomeres(端粒酶)

Materials and Reagents

  1. Cells
  2. 4% PFA
  3. Methanol/acetone 1:1
  4. TritonX100
  5. Primary antibody : 53BP1 #NB 100-304 rabbit from Novus
  6. Second antibody: goat anti-rabbit Alexa Fluor® 488 Dye
  7. PBS
  8. Glycine
  9. Fish gelatin (Sigma-Aldrich, catalog number : G7041 )
  10. BSA
  11. Formamide
  12. Tris HCl, pH 7.4
  13. Telomeric PNA probe (TelC-Cy3 from PANAGENE, catalog number: F1002-5 )
  14. DAPI
  15. Mowiol 4-88 reagent (Calbiochem®)
  16. PBG (see Recipes)
  17. Hybridization mixture (see Recipes)
  18. Blocking reagent (Roche Diagnostics, catalog number: 11096176001 ) (see Recipes)
  19. Wash solution I (see Recipes)
  20. Wash solution II (see Recipes)

Equipment

  1. Glass coverslips
  2. 12 multiwell plate
  3. Metal thermoblock
  4. Humidified chamber

Procedure

  1. Grow cells on glass coverslips (e.g. BJ normal human fibroblasts).
  2. Transfer the coverslip to a 12 multiwell plate.
  3. Wash briefly with 1x PBS.
  4. Fix with either 4% PFA, 10 min, RT or methanol/acetone 1:1, 2 min, RT (it depends on the antibody, does not affect the FISH signal; use methanol/acetone for 53BP1 staining).
  5. Wash with 1x PBS, 3 times, 5 min.
  6. Only for PFA-fixed cells, incubate with 0.2% TritonX100 in PBS, 10 min, then wash with 1x PBS, 3 times, 5 min.
  7. Block with 1x PBG, 1 h, RT.
  8. Incubate with primary antibody diluted in 1x PBG, 50 μl for each coverslip. Incubation time depends on the antibody, most work in 1 h, RT, or overnight at 4 °C. (For 53BP1 dilute 1:200 and incubate 1 h at RT).
  9. Wash with 1x PBG, 3 times, 5 min.
  10. Incubate with secondary antibody diluted in 1x PBG, 45 min, RT.
  11. Wash with 1x PBG, twice, 5 min.
  12. Wash with 1x PBS, twice, 5 min.
  13. Re-fix cells with PFA 4% + triton 0.1%, 10 min RT (use PFA also if you have previously fixed cells with methanol/acetone).
  14. Incubate with glycine 10 mM in H2O, 30 min, RT.
  15. Wash with 1x PBS, 3 times, 5 min.
  16. Prepare the hybridization mixture and put 20 μl on a glass slide for each coverslip.
  17. Transfer the coverslip carefully on the drop without making bubbles.
  18. Put the slide directly on a metal thermoblock at 80 °C, 5 min.
  19. Hybridize in a humidified chamber, 2 h, RT.
  20. Remove coverslip from the slide and put it back in the 12 wells plate.
  21. Wash with Wash solution I, twice, 15 min.
  22. Wash with Wash solution II, 3 times, 5 min.
  23. Incubate with DAPI, 2 min, RT.
  24. Wash briefly with 1x PBS.
  25. Mount with mowiol.
  26. Store the slides at 4 °C for short time storage (2 weeks) or at -20 °C. It is recommended to analyze the fluorescence as soon as possible to avoid fluorophore fading.


    Figure 1. An image of ImmunoFish stained human fibroblasts cells. DAPI is in blue, 53BP1is in green and telomeric PNA probe is in red.

Recipes

  1. 10x PBG (prepare 5 ml aliquotes and store them in 50 ml tubes at -20 °C, the day of immunoFISH dilute them in 1x PBS)
    Fish gelatin
    2%
    BSA
    5%
    1x PBS
    to volume
  2. Hybridization mixture (always prepare fresh)
    Formamide
    70%
    Blocking reagent
    1x
    Tris HCl pH 7.4
    10 mM
    Telomeric PNA probe
    0.5 μM
    H2O
    to volume
  3. 10x Blocking reagent
    Prepare small aliquots and store them at -20 °C
  4. Wash Solution I (250 ml) (always prepare fresh)
    Formamide
    175 ml
    BSA 10%
    2.5 ml
    Tris HCl 1 M pH 7.4
    2.5 ml
    H2O
    to volume
  5. Wash Solution II (350 ml) (always prepare fresh)
    Tris HCl 1 M pH 7.4
    35 ml
    NaCl 5 M
    10.5 ml
    Tween 20 10%
    2.5 ml
    H2O
    to volume

Acknowledgments

The F.d’A.d.F. laboratory is supported by FIRC (Fondazione Italiana per la Ricerca sul Cancro), AIRC (Associazione Italiana per la Ricerca sul Cancro), European Union (GENINCA, contract number 202230), HFSP (Human Frontier Science Program), AICR (Association for International Cancer Research), EMBO Young Investigator Program and Telethon.

References

  1. Fumagalli, M., Rossiello, F., Clerici, M., Barozzi, S., Cittaro, D., Kaplunov, J. M., Bucci, G., Dobreva, M., Matti, V. and Beausejour, C. M. (2012). Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation. Nat Cell Biol 14(4): 355-365.

简介

该方案针对贴壁培养的哺乳动物细胞的免疫FISH染色进行优化。 它结合DNA损伤应答因子(例如53BP1)的免疫荧光和针对端粒DNA的FISH。

关键字:immunofish, DNA损伤反应, 端粒酶

材料和试剂

  1. 单元格
  2. 4%PFA
  3. 甲醇/丙酮1:1
  4. TritonX100
  5. 一抗:来自Novus的53BP1 #NB 100-304兔子
  6. 第二抗体:山羊抗兔Alexa Fluor 488染料
  7. PBS
  8. 甘氨酸
  9. 鱼明胶(Sigma-Aldrich,目录号:G7041)
  10. BSA
  11. 甲酰胺
  12. Tris HCl,pH 7.4
  13. 端粒PNA探针(来自PANAGENE的TelC-Cy3,目录号:F1002-5)
  14. DAPI
  15. Mowiol 4-88试剂(Calbiochem )
  16. PBG(参见配方)
  17. 杂交混合物(参见配方)
  18. 封闭试剂(Roche Diagnostics,目录号:11096176001)(参见配方)
  19. 洗涤溶液I(参见配方)
  20. 洗液II(参见配方)

设备

  1. 玻璃盖片
  2. 12多孔板
  3. 金属热块
  4. 加湿室

程序

  1. 在玻璃盖玻片上生长细胞(例如 BJ正常人成纤维细胞)。
  2. 将盖玻片转移到12个多孔板。
  3. 用1x PBS简单冲洗。
  4. 用4%PFA,10分钟,RT或甲醇/丙酮1:1,2分钟,RT固定(取决于抗体,不影响FISH信号;使用甲醇/丙酮进行53BP1染色)。
  5. 用1×PBS清洗,3次,5分钟
  6. 仅对于PFA固定的细胞,用0.2%TritonX100在PBS中温育10分钟,然后用1x PBS洗涤3次,5分钟。
  7. 使用1x PBG,1小时,RT阻止。
  8. 孵育与一级PBG稀释的一抗,每个盖玻片50微升。 孵育时间取决于抗体,大多数工作在1小时,RT,或在4℃过夜。 (对于53BP1稀释1:200并在室温下孵育1小时)
  9. 用1×PBG,3次,5分钟洗涤
  10. 与在1x PBG中稀释的第二抗体孵育,45分钟,RT
  11. 用1x PBG洗涤两次,每次5分钟。
  12. 用1x PBS洗涤两次,每次5分钟。
  13. 用PFA 4%+ triton 0.1%,10分钟RT(如果你以前用甲醇/丙酮固定细胞,也用PFA)重新固定细胞。
  14. 与10mM甘氨酸在H 2 O中孵育30分钟,RT
  15. 用1×PBS清洗,3次,5分钟
  16. 准备杂交混合物,并将20μl放在载玻片上的每个盖玻片
  17. 小心地将盖玻片转移到滴上而不产生气泡。
  18. 将载玻片直接放在金属加热块上,在80℃,5分钟
  19. 在加湿室中杂交,2小时,RT
  20. 从幻灯片中取出盖玻片,并将其放回12孔板
  21. 用洗涤溶液I洗涤两次,15分钟。
  22. 用洗涤溶液II洗涤,3次,5分钟
  23. 用DAPI孵育,2分钟,RT
  24. 用1x PBS简单冲洗。
  25. 用mowiol装载。
  26. 将载玻片存储在4°C短时储存(2周)或-20°C。 建议尽快分析荧光,以避免荧光团褪色

    图1. ImmunoFish染色的人成纤维细胞的图像。 DAPI为蓝色,53BP1为绿色,端粒PNA探针为红色。

食谱

  1. 10×PBG(制备5ml等分试样并将其在-20℃下储存在50ml管中,免疫FISH的天将其稀释在1×PBS中)
    鱼明胶
    2%
    BSA
    5%
    1x PBS
    到卷
  2. 杂交混合物(总是准备新鲜)
    甲酰胺
    70%
    封闭试剂
    1x
    Tris HCl pH7.4
    10 mM
    端粒PNA探针
    0.5μM
    H sub 2 O
    到卷
  3. 10x封闭试剂
    准备小份,并将其存储在-20°C
  4. 洗涤溶液I(250ml)(总是准备新鲜)
    甲酰胺
    175 ml
    BSA 10%
    2.5 ml
    Tris HCl 1M pH7.4
    2.5 ml
    H sub 2 O
    到卷
  5. 洗涤溶液II(350ml)(总是准备新鲜)
    Tris HCl 1M pH7.4
    35 ml
    NaCl 5×m/v 10.5 ml
    吐温20 10%
    2.5 ml
    H sub 2 O
    到卷

致谢

F.d'A.d.F。 实验室由FIRC(意大利人拉科尔卡),AIRC(意大利人协会),欧盟(GENINCA,合同号202230),HFSP(人类前沿科学计划),AICR(国际癌症协会 研究),EMBO青年研究者计划和Telethon。

参考文献

  1. Fumagalli,M.,Rossiello,F.,Clerici,M.,Barozzi,S.,Cittaro,D.,Kaplunov,JM,Bucci,G.,Dobreva,M.,Matti,V。和Beausejour, 。 端粒DNA损伤是不可挽回的,并导致持续的DNA损伤反应激活。 Nat Cell Biol 14(4):355-365
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rossiello, F., Fumagalli, M. and di Fagagna, F. d. (2013). ImmunoFISH for Adherent Cultured Mammalian Cells. Bio-protocol 3(24): e999. DOI: 10.21769/BioProtoc.999.
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