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In this protocol, different types of adherent cells are fixed to coverslips by 4% paraformaldehyde. Target proteins are then stained by specific antibodies and fluorescent 2nd antibodies. This protocol therefore provides a method for immunostaining of adherent cells.

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Immunofluorescence (Indirect Staining) Protocol for Adherent Cells
贴壁细胞免疫荧光(间接染色) 方法

细胞生物学 > 细胞成像 > 荧光
作者: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
Vol 2, Iss 3, 2/5/2012, 13656 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.50

[Abstract] In this protocol, different types of adherent cells are fixed to coverslips by 4% paraformaldehyde. Target proteins are then stained by specific antibodies and fluorescent 2nd antibodies. This protocol therefore provides a method for immunostaining of adherent cells.
Keywords: Immunofluorescence(免疫荧光), Cell(细胞), Adherent(贴壁)

[Abstract] 在该协议中,不同类型的粘附细胞通过4%多聚甲醛固定到盖玻片上。 然后通过特异性抗体和荧光2 nd抗体对靶蛋白进行染色。 因此该方案提供了用于贴壁细胞的免疫染色的方法。

Materials and Reagents

  1. Raw264.7, MCF-7 or Hela cells
  2. DPBS (Life Technologies, InvitrogenTM, catalog number: 14190-250 )
  3. FBS (Atlanta Biologicals, catalog number: S11110H )
  4. Anti-fade mounting medium: e.g. ProLong Gold Antifade Mountant (Life Technologies, InvitrogenTM, catalog number: P10144 ) with DAPI (if nuclear staining is needed) or without DAPI
  5. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
  6. General chemicals (Sigma-Aldrich)
  7. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  8. Tween 20 (Sigma-Aldrich, catalog number: P2287 )
  9. 4% paraformaldehyde-freshly-prepared (5 ml) (see Recipes)
  10. Antibodies (see Recipes)

Equipment

  1. Round cover slips (Thermo Fisher Scientific)
  2. 24-well plate
  3. Fluorescence microscope

Procedures

  1. Grow cells on round cover slips in 24-well plates to 30-60% confluence. Do not overgrow because it will be difficult to distinguish cell components when stained.
  2. Wash cells with 500 μl/well DPBS twice, then add 250 μl/well 4% paraformaldehyde for 15 min at room temperature (RT).
  3. Remove paraformaldehyde, wash the fixed cells with 500 μl/well PBS + 3% FBS for 3 times.
  4. Permeabilize cells with 250 μl/well DPBS + 0.2% Triton X-100 for 5 min, then wash with 500 μl/well DPBS + 3% FBS for 3 times.
  5. Block with 500 μl/well DPBS + 3% FBS + 0.5% Tween 20 for 1 h at RT.
  6. Remove the blocking buffer, add 250 μl/well primary antibodies and incubate at RT for 1 h.
  7. Remove the antibodies, wash with 500 μl/well DPBS + 3% FBS in 5 min for 3 times.
  8. Add 250 μl/well fluorescent secondary antibodies and incubate at RT for 30 min.
  9. Remove the antibodies, wash with 500 μl/well DPBS + 3% FBS in 5 min for 3 times.
  10. Place a small drop of anti-fade reagent on a glass slide, then get the cover slip from the well and put it face down on the drop, push it tightly and attach to the glass slide.
  11. Leave the slide in the dark for 5 min to let it dry.
  12. The slide now it is ready to observe under a fluorescence microscope.

Recipes

  1. 4% paraformaldehyde-freshly-prepared (5 ml)
    0.2 g paraformaldehyde powder + 5 ml DPBS + 50 μl 1 N NaOH
    Incubate at 65 °C
    Vortex several times to dissolved completely
    Cool at room temperature then add 4 μl HCl
    Mixed completely
  2. Antibodies
    Diluted in DPBS + 3% FBS + 0.5 Tween 20
    Primary antibodies: 1:100-1:500 (depends on individual antibodies)
    Fluorescent secondary antibodies: 1:800

Acknowledgments

This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. This protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA [Chen et al. (unpublished)].

References

  1. Agrawal, S., van Dooren, G. G., Beatty, W. L. and Striepen, B. (2009). Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins. J Biol Chem 284(48): 33683-33691.

材料和试剂

  1. Raw264.7,MCF-7或Hela细胞
  2. DPBS(Life Technologies,Invitrogen TM,目录号:14190-250)
  3. FBS(Atlanta Biologicals,目录号:S11110H)
  4. 抗褪色封固介质:使用DAPI(如果需要核染色)或不使用DAPI 的ProLong Gold Antifade Mountant(Life Technologies,Invitrogen TM ,目录号:P10144) br />
  5. 多聚甲醛(Sigma-Aldrich,目录号:158127)
  6. 一般化学品(Sigma-Aldrich)
  7. Triton X-100(Sigma-Aldrich,目录号:T8787)
  8. 吐温20(Sigma-Aldrich,目录号:P2287)
  9. 4%多聚甲醛新鲜制备(5ml)(参见配方)
  10. 抗体(参见配方)

设备

  1. 圆形盖玻片(Thermo Fisher Scientific)
  2. 24孔板
  3. 荧光显微镜

程序

  1. 在24孔板中的圆盖玻片上生长细胞至30-60%汇合。 不要过度生长,因为在染色时很难区分细胞组分
  2. 用500μl/孔DPBS洗涤细胞两次,然后在室温(RT)下加入250μl/孔4%多聚甲醛15分钟。
  3. 去除多聚甲醛,用500μl/孔PBS + 3%FBS洗涤固定的细胞3次
  4. 用250μl/孔DPBS + 0.2%Triton X-100透化细胞5分钟,然后用500μl/孔DPBS + 3%FBS洗涤3次。
  5. 用500μl/孔DPBS + 3%FBS + 0.5%Tween 20在室温封闭1小时
  6. 取出封闭缓冲液,加入250μl/孔一抗,室温孵育1小时
  7. 取出抗体,用500μl/孔DPBS + 3%FBS在5分钟内洗涤3次
  8. 加入250μl/孔荧光第二抗体,并在室温下孵育30分钟
  9. 取出抗体,用500μl/孔DPBS + 3%FBS在5分钟内洗涤3次
  10. 在玻璃载玻片上放一小滴防褪色试剂,然后从孔中取出盖玻片,并将其面朝下放在玻璃片上,将其紧紧压在玻片上。
  11. 离开幻灯片在黑暗中5分钟,让它干燥。
  12. 幻灯片现在准备在荧光显微镜下观察。

食谱

  1. 4%多聚甲醛新鲜制备(5ml) 0.2g多聚甲醛粉末+ 5ml DPBS +50μl1N NaOH
    在65℃孵育
    涡旋几次以完全溶解
    室温下冷却,然后加入4μlHCl
    完全混合
  2. 抗体
    在DPBS + 3%FBS + 0.5Tween 20中稀释 初级抗体:1:100-1:500(取决于单个抗体)
    荧光二抗:1:800

致谢

这项工作由杭州高新区5050项目,杭州市海外退学基金资助,浙江省ZJ1000项目资助。 这个协议是在科恩实验室,遗传学,斯坦福大学开发的 University,CA,USA [Chen 等(未公开)]。

参考文献

  1. Agrawal,S.,van Dooren,G.G.,Beatty,W.L.and Striepen,B。(2009)。 基因证据表明endosymbiont衍生的内质网相关蛋白降解(ERAD)系统在进口的功能 美国生物化学杂志  284(48):33683-33691。
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How to cite this protocol: Chen, R. (2012). Immunofluorescence (Indirect Staining) Protocol for Adherent Cells. Bio-protocol 2(3): e50. DOI: 10.21769/BioProtoc.50; Full Text



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