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)
Materials and Reagents
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Raw264.7, MCF-7 or Hela cells
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DPBS (Life Technologies, InvitrogenTM, catalog number: 14190-250 )
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FBS (Atlanta Biologicals, catalog number: S11110H )
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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
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Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
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General chemicals (Sigma-Aldrich)
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Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
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Tween 20 (Sigma-Aldrich, catalog number: P2287 )
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4% paraformaldehyde-freshly-prepared (5 ml) (see Recipes)
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Antibodies (see Recipes)
Equipment
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Round cover slips (Thermo Fisher Scientific)
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24-well plate
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Fluorescence microscope
Procedures
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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.
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Wash cells with 500 μl/well DPBS twice, then add 250 μl/well 4% paraformaldehyde for 15 min at room temperature (RT).
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Remove paraformaldehyde, wash the fixed cells with 500 μl/well PBS + 3% FBS for 3 times.
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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.
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Block with 500 μl/well DPBS + 3% FBS + 0.5% Tween 20 for 1 h at RT.
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Remove the blocking buffer, add 250 μl/well primary antibodies and incubate at RT for 1 h.
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Remove the antibodies, wash with 500 μl/well DPBS + 3% FBS in 5 min for 3 times.
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Add 250 μl/well fluorescent secondary antibodies and incubate at RT for 30 min.
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Remove the antibodies, wash with 500 μl/well DPBS + 3% FBS in 5 min for 3 times.
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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.
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Leave the slide in the dark for 5 min to let it dry.
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The slide now it is ready to observe under a fluorescence microscope.
Recipes
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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
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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
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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.
简介
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。
关键字:免疫荧光, 细胞, 贴壁
材料和试剂
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Raw264.7,MCF-7或Hela细胞
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DPBS(Life Technologies,Invitrogen TM,目录号:14190-250)
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FBS(Atlanta Biologicals,目录号:S11110H)
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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 />
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多聚甲醛(Sigma-Aldrich,目录号:158127)
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一般化学品(Sigma-Aldrich)
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Triton X-100(Sigma-Aldrich,目录号:T8787)
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吐温20(Sigma-Aldrich,目录号:P2287)
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4%多聚甲醛新鲜制备(5ml)(参见配方)
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抗体(参见配方)
设备
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圆形盖玻片(Thermo Fisher Scientific)
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24孔板
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荧光显微镜
程序
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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
- 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。
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去除多聚甲醛,用500μl/孔PBS + 3%FBS洗涤固定的细胞3次
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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。
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用500μl/孔DPBS + 3%FBS + 0.5%Tween 20在室温封闭1小时
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取出封闭缓冲液,加入250μl/孔一抗,室温孵育1小时
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取出抗体,用500μl/孔DPBS + 3%FBS在5分钟内洗涤3次
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加入250μl/孔荧光第二抗体,并在室温下孵育30分钟
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取出抗体,用500μl/孔DPBS + 3%FBS在5分钟内洗涤3次
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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。
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离开幻灯片在黑暗中5分钟,让它干燥。
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幻灯片现在准备在荧光显微镜下观察。
食谱
- 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
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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
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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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