欢迎您, 登录 | 注册

首页 | English

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

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, 12406 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] 用4%的多聚甲醛使细胞粘附,通过特异性的抗体和荧光标记的二抗对目的蛋白进行染色。

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.     圆形盖玻片 (Fisher)

2.     DPBS (Invitrogen)

3.     FBS (Atlanta)

4.     抗褪色封固剂: ProLong Gold antifade reagent from Invitrogen, with DAPI (if need nuclear staining) or without DAPI.

5.     多聚甲醛 (Sigma)

6.     常用化学试剂 (Sigma)

 

实验步骤

1.  让细胞生长在放入24孔细胞培养板中的圆形盖玻片上,使其融合度达到30-60%,不要使细胞长过,如果长过会在染色时难以区别细胞的组分。

2.  每孔用500ulDPBS洗细胞2次,之后每孔加250ul 4% 多聚甲醛,室温固定15min

3.  去除多聚甲醛,每孔用500 ul3%FBS PBS洗细胞3

4.  每孔用250ul0.2%Triton X-100 DPBS透化细胞5 min,每孔用500 ul3%FBS PBS洗细胞3

5.  每孔加入500ulDPBS(含3%FBS0.5% Tween20),室温封闭1小时。

6.  去除封闭液,每孔加250 ul一抗,室温孵育1小时

7.  去除抗体,每孔用500 ul3%FBS PBS洗细胞3次,每次5min

8.  每孔加250 ul 荧光二抗,室温孵育30 min.

9.  去除二抗, 每孔用500 ul3%FBS PBS洗细胞3次,每次5min

7.  载玻片上滴一小滴抗褪色剂,将盖玻片从孔中取出,面朝下盖到抗褪色剂上,使其轻轻接触到载玻片上。将玻片置于暗处5min使其晾干,此时即可在荧光显微镜下进行观察。

 

配液

1.  4%多聚甲醛(现用现配)(5ml): 0.2g 多聚甲醛粉末+  5ml DPBS + 50ul 1MNaOH, 65孵育, 反复涡旋使其完全溶解, 室温冷却, 4ul HCl, 充分混匀

2.     抗体, DPBS (含 3%FBS0.5 Tween20)稀释

一抗:  1:100-1:500(不同的抗体稀释比例不同)

荧光二抗:  1:800.

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。

X


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



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册