ImmunoFISH for Adherent Cultured Mammalian Cells
附着培养哺乳动物细胞的ImmunoFISH试验
发布: 2013年12月20日第3卷第24期 DOI: 10.21769/BioProtoc.999 浏览次数: 11720
评审: Lin Fang
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参见作者原研究论文
The authors used this protocol in:
Apr 2012
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
- Cells
- 4% PFA
- Methanol/acetone 1:1
- TritonX100
- Primary antibody : 53BP1 #NB 100-304 rabbit from Novus
- Second antibody: goat anti-rabbit Alexa Fluor® 488 Dye
- PBS
- Glycine
- Fish gelatin (Sigma-Aldrich, catalog number : G7041 )
- BSA
- Formamide
- Tris HCl, pH 7.4
- Telomeric PNA probe (TelC-Cy3 from PANAGENE, catalog number: F1002-5 )
- DAPI
- Mowiol 4-88 reagent (Calbiochem®)
- PBG (see Recipes)
- Hybridization mixture (see Recipes)
- Blocking reagent (Roche Diagnostics, catalog number: 11096176001 ) (see Recipes)
- Wash solution I (see Recipes)
- Wash solution II (see Recipes)
Equipment
- Glass coverslips
- 12 multiwell plate
- Metal thermoblock
- Humidified chamber
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
细胞生物学 > 细胞结构 > 染色体
细胞生物学 > 细胞成像 > 荧光
生物化学 > 蛋白质 > 免疫检测
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