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Indirect Immunofluorescence Assay in Chlamydomonas reinhardtii
莱茵衣藻的间接免疫荧光测定   

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Abstract

Determining the protein localization is essential to elucidate its in vivo function. Fluorescence-tagged proteins are widely used for it, but it is sometimes difficult to express tagged proteins in Chlamydomonas. Alternatively, indirect immunofluorescence assay is also one of the widely used methods and many reports determining the localization of Chlamydomonas proteins using this method are published. Here, we introduce a protocol of indirect immunofluorescence assay adapted from our papers reporting LCIB (CO2-recycling factor in the vicinity of pyrenoid; Yamano et al., 2010), LCI1 (plasma membrane-localized inorganic carbon transporter; Ohnishi et al., 2010), HLA3 (plasma membrane-localized ABC-type bicarbonate transporter; Yamano et al., 2015), and LCIA (chloroplast envelope anion channel; Yamano et al., 2015) in Chlamydomonas reinhardtii. The protocol described here could be useful for observing the protein of interest in other algae cells.

Keywords: Chloroplast membrane(叶绿体膜), Bicarbonate transporter(碳酸氢盐运输), Micro-algae(微型藻类), Photosynthesis(光合作用), Pyrenoid(蛋白核)

Materials and Reagents

  1. Poly-L-lysine-treated glass slide (Poly-Prep Slides) (Sigma-Aldrich, catalog number: P0425-72EA )
  2. Coverslips (Matsunami Glass, catalog number: C218181 )
  3. Dako Pen (Dako, catalog number: S2002 )
  4. Plastic box (9 cm x 19 cm x 4 cm)
    Note: In this experiment, but any size is OK.
  5. Kimwipes® paper (Kimberly Clark)
  6. Chlamydomonas cells of interest
  7. Hematocytometer (Erma, catalog number: 03-303-6 )
  8. Phosphate-buffered Saline (PBS)
  9. Tween-20 (Santa Cruz, catalog number: sc-29113 )
  10. PBS-T (PBS with 0.1% Tween-20)
  11. Paraformaldehyde (Nacalai Tesque, catalog number: 26126-54 )
  12. Methanol (Nacalai Tesque, catalog number: 21915-64 )
  13. Globulin-free Bovine Serum Albumin (BSA) (Nacalai Tesque, catalog number: 01281-26 )
  14. Affinity-purified rabbit primary antibody against LCIB (Yamano et al., 2010)
  15. Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (Thermo Fisher, catalog number: A-11001 )
  16. Mounting medium (Vectorshield, catalog number: H-1300 )
  17. 4% formaldehyde (see Recipes)
  18. PBS (see Recipes)
  19. PBS-T (see Recipes)
  20. Blocking solution (see Recipes)

Equipment

  1. Coplin jar (50 ml size)
  2. Fluorescence microscopy Axioscope2 (Zeiss) with a specific filter set (excitation bandpass
    480/40 and emission band pass 527/30) or laser scanning confocal microscopy TCS SP8
    (Leica) with a 488 nm laser line

Note: these were the instruments used throughout our experiments, but any company is OK.

Procedure

  1. Chill 100% methanol in a Coplin jar at -20 °C for at least 4 h.
  2. Harvest 50 ml of Chlamydomonas cells at the log growth phase by centrifugation at 600 x g for 5 min. This and subsequent steps are performed at room temperature except for step 10.
    Notes:
    1. Chlamydomonas culture media recipes are available at http://www.chlamy.org/media.html.
    2. 50 ml of cultured cells grown to 1-2 x 106 cells ml–1 is adequate.
  3. Resuspend the cells in small volumes (1-2 ml) of PBS, count the cell number by hematocytometer, and adjust the cell density at 1x107 cells ml–1 using PBS.
  4. Draw three circles on a Poly-L-lysine-treated glass slide with Dako Pen to provide a barrier to liquids such as blocking solution, antibody solution, and washing buffer (Figure 1A). Dry the circles at room temperature.
  5. Put 100 µl of Chlamydomonas cell suspension in each circle and let them air-dry for 5 min to fix the cells to the glass.
    Note: If you let them sit longer, more Chlamydomonas cells will adhere, but the ends of the flagella of cells start to curl up.
  6. Permeabllize the cells by immersing the glass slide into a Coplin jar filled with PBS-T for 10 min (Figure 1B).
  7. Prepare a clean plastic box with a few damp Kimwipes® paper at the bottom to keep the air humid.
  8. Place the glass slide on a cap of plastic tube placed in the plastic box (Figure 1C) and fix the proteins within cells by applying 100 µl of PBS containing 4% (w/v) formaldehyde to the cells for 20 min. Make sure that the solution is within the circles and the entire cells are covered by the PBS solution.


    Figure 1. Devices for indirect immunofluorescence assay. A. Example of circles drawn by Dako Pen on a Poly-L-lysine-treated glass slide. B. Immersed glass slide in a Coplin jar filled with PBS-T. C. Humid plastic box with a few damp Kimwipes® papers and a glass slide on a cap of plastic tube.

  9. Wash the cells two times by immersing the glass slide into a Coplin jar filled with PBS for 5 min.
    Note: You do not need to shake the slides or jars during washing.
  10. Fix the cells by immersing the glass slide into a Coplin jar filled with pre-chilled 100% methanol at -20 ˚C for 20 min. Because chlorophyll will be solubilized, the methanol becomes green.
    Note: Because the temperature of the chilled methanol is important in the fixation, it is recommended to keep the methanol in a freezer at -20 ˚C overnight before use.
  11. Rehydrate the cells by immersing the glass slide into a Coplin jar filled with PBS for 5 min.
  12. Pour off the PBS and repeat the rehydration step 11 two times. If you perform time-course experiments, you can let the glass slide sit in PBS for several hours until you are ready.
  13. Take out the glass slide from the Coplin jar and let it air-dry for 5 min.
  14. Block the cells on the glass slide by applying 100 µl of blocking solution for 1 h in the humid plastic box.
  15. Wash the cells two times by immersing the glass slide into a Coplin jar filled with PBS for 5 min.
    Note: You do not need to shake the slides or jars during washing.
  16. Take out the glass slide from the Coplin jar and let it air-dry for 5 min.
  17. Apply 50 µl of PBS-T containing the primary antibody at the dilution rate of 1:500 and incubate the cells for 1 h in the humid plastic box.
  18. Wash the cells six times by immersing the glass slide into a Coplin jar filled with PBS-T for 5 min.
  19. Apply 50 µl of PBS-T containing secondary antibody at the dilution rate of 1:500 and incubate the cells for 1 h in the humid plastic box. Keep in the dark.
  20. Wash the cells six times by immersing the glass slide into a Coplin jar filled with PBS-T for 5 min.
  21. Apply 15 µl of mounting solution to the cells. Make sure that no air bubbles are formed.
  22. Cells are now ready for observation by fluorescent microscopy with a specific filter set (excitation bandpass 480/40 and emission bandpass 527/30) or laser scanning confocal microscopy with a 488 nm laser line. Glass slide can be stored for a week at 4 °C in the dark.


    Figure 2. Actual cell images and fluorescence signals of indirect immunofluorescence assay. A. Fixed cells on the glass slide after step 5. Scale bar, 10 µm. B. Fixed cells on the glass slide after step 10. Chlorophyll pigments are extracted and the color of the cells turn transparent. Scale bar, 10 µm. C. Differential interference contrast image of Chlamydomonas cell. Arrowhead indicates the pyrenoid structure where CO2-fixation enzyme Rubisco is enriched. Scale bar, 5 µm. D. Indirect immunofluorescence signals derived from anti LCIB-antibody. LCIB is detected as a ring-like structure around the pyrenoid as shown in the previous report (Yamano et al., 2010). Scale bar, 5 µm.

Notes

We highly recommend that user also performs the same experiment using a target gene mutant as a negative control to distinguish true signals from artifact.

Recipes

  1. 4% formaldehyde
    1. Heat 80 ml of PBS to approximately 60 °C on a stir plate.
    2. Add 4 g of paraformaldehyde and slowly raise the pH by adding 1 N NaOH until the solution becomes clear.
    3. After dissolving, cool and filter the solution.
    4. Fill up the volume to 100 ml with PBS and adjust the pH with diluted HCl to approximately 6.9.
    5. Aliquoted solutions can be kept at -20 °C for a month.
  2. PBS
    Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 900 ml of distilled H2O. Adjust the pH to 7.4 with HCl and add H2O to 1L.
  3. PBS-T
    Add 0.1% Tween-20 (v/v) to PBS.
  4. Blocking solution
    5% (w/v) globulin-free BSA in PBS buffer

Acknowledgments

We thank Lianyong Wang for technical assistance. This work was supported by the Japan Society for the Promotion of Science KAKENHI Grants 25120714 (to H.F.) and 25840109 (to T.Y.) and the Japan Science and Technology Agency Advanced Low Carbon Technology Research and Development Program.

References

  1. Ohnishi, N., Mukherjee, B., Tsujikawa, T., Yanase, M., Nakano, H., Moroney, J. V. and Fukuzawa, H. (2010). Expression of a low CO(2)-inducible protein, LCI1, increases inorganic carbon uptake in the green alga Chlamydomonas reinhardtii. Plant Cell 22(9): 3105-3117.
  2. Shakes, D. C., Miller, D. M., 3rd and Nonet, M. L. (2012). Immunofluorescence microscopy. Methods Cell Biol 107: 35-66.
  3. Yamano, T., Tsujikawa, T., Hatano, K., Ozawa, S., Takahashi, Y. and Fukuzawa, H. (2010). Light and low-CO2-dependent LCIB-LCIC complex localization in the chloroplast supports the carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Cell Physiol 51(9): 1453-1468.
  4. Yamano, T., Sato, E., Iguchi, H., Fukuda, Y. and Fukuzawa, H. (2015). Characterization of cooperative bicarbonate uptake into chloroplast stroma in the green alga Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 112(23): 7315-7320.

简介

确定蛋白质定位是阐明其体内功能所必需的。荧光标记的蛋白质被广泛使用,但有时难以在衣原体中表达标记的蛋白质。或者,间接免疫荧光测定也是广泛使用的方法之一,并且公开了使用该方法确定衣藻蛋白定位的许多报道。在这里,我们介绍间接免疫荧光测定方案改编自我们的论文报告LCIB(在芘类化合物附近的CO 2 - 再循环因子; Yamano等人,2010), LCI1(质膜定位的无机碳转运蛋白; Ohnishi等人,2010),HLA3(浆膜定位的ABC-型碳酸氢盐转运蛋白; Yamano等人,2015) )和LCIA(叶绿体包膜阴离子通道; Yamano等人,2015)在莱茵衣藻(Chlamydomonas reinhardtii)中。这里描述的协议可以用于观察其他藻类细胞感兴趣的蛋白质。

关键字:叶绿体膜, 碳酸氢盐运输, 微型藻类, 光合作用, 蛋白核

材料和试剂

  1. 聚-L-赖氨酸处理的载玻片(Poly-Prep载玻片)(Sigma-Aldrich,目录号:P0425-72EA)
  2. 盖玻片(Matsunami Glass,目录号:C218181)
  3. Dako Pen(Dako,目录号:S2002)
  4. 塑料盒(9厘米×19厘米×4厘米)
    注意:在此实验中,任何大小都可以。
  5. Kimwipes ?纸(Kimberly Clark)
  6. 感兴趣的细胞
  7. 血细胞计数器(Erma,目录号:03-303-6)
  8. 磷酸盐缓冲盐水(PBS)
  9. Tween-20(Santa Cruz,目录号:sc-29113)
  10. PBS-T(含0.1%Tween-20的PBS)
  11. 多聚甲醛(Nacalai Tesque,目录号:26126-54)
  12. 甲醇(Nacalai Tesque,目录号:21915-64)
  13. 无球蛋白牛血清白蛋白(BSA)(Nacalai Tesque,目录号:01281-26)
  14. 亲和纯化的针对LCIB的兔一抗(Yamano等人,2010)
  15. 山羊抗兔IgG(H + L)第二抗体,Alexa Fluor?488缀合物(Thermo Fisher,目录号:A-11001)
  16. 安装介质(Vectorshield,目录号:H-1300)
  17. 4%甲醛(见配方)
  18. PBS(请参阅配方)
  19. PBS-T(参见配方)
  20. 阻塞溶液(参见配方)

设备

  1. Coplin罐(50 ml尺寸)
  2. 荧光显微镜Axioscope2(Zeiss)具有特定的滤光片组(激发带通
    480/40和发射带通527/30)或激光扫描共焦显微镜TCS SP8
    (Leica),具有488nm激光线

注意:这些是我们实验中使用的仪器,但任何公司都可以。

程序

  1. 在-20℃下在coplin罐中冷却100%甲醇至少4小时
  2. 通过在600×g离心5分钟,在对数生长期收获50ml的莱茵衣藻细胞。该步骤和随后的步骤在室温下进行,除了步骤10之外 注意:
    1. 衣藻培养基配方可在 http ://www.chlamy.org/media.html
    2. 将培养细胞生长至1-2×10 6个 6 细胞的50ml培养细胞ml 1 已足够。
  3. 在小体积(1-2ml)的PBS中重悬细胞,通过血细胞计数器计数细胞数,并使用PBS调节1×10 7个细胞ml -1 -1的细胞密度。
  4. 用Dako Pen在聚-L-赖氨酸处理的载玻片上绘制三个圆圈,以提供对液体如阻断溶液,抗体溶液和洗涤缓冲液的屏障(图1A)。在室温下干燥圆圈。
  5. 将100μl的衣藻细胞悬浮液置于每个圈中,并让它们空气干燥5分钟以将细胞固定在玻璃杯上。
    注意:如果你让他们坐得更长,更多的衣藻细胞会粘附,但细胞鞭毛的末端开始卷曲。
  6. 通过将载玻片浸入装有PBS-T的Coplin罐中10分钟,使细胞可渗透(图1B)。
  7. 准备一个干净的塑料盒,底部有少量潮湿的Kimwipes ?纸,以保持空气湿润。
  8. 放置玻璃载玻片上的塑料管放置在塑料盒(图1C),并固定蛋白质在细胞内,通过应用100微升含4%(w/v)甲醛的PBS的细胞20分钟。确保溶液在圆内,并且整个细胞被PBS溶液覆盖。


    图1.用于间接免疫荧光测定的装置。 A.由Dako Pen在聚-L-赖氨酸处理的载玻片上绘制的圆圈的实例。 B.浸没的玻璃载玻片在装有PBS-T的Coplin罐中。 C.湿润的塑料盒,带有少量潮湿的Kimwipes ?纸和玻璃片在塑料管的帽子上。

  9. 通过将载玻片浸入装有PBS的Coplin罐中5分钟来洗涤细胞两次 注意:您不需要在洗涤期间摇动幻灯片或瓶子。
  10. 通过将载玻片浸入装有预冷的100%甲醇的coplin罐中在-20℃下固定细胞20分钟来固定细胞。因为叶绿素会溶解,甲醇变成绿色 注意:因为冷冻甲醇的温度在固定中很重要,建议在使用前将甲醇保存在-20℃的冰箱中过夜。
  11. 通过将载玻片浸入装有PBS的Coplin罐中5分钟来再水合细胞
  12. 倒出PBS并重复水化步骤11两次。如果你进行时间实验,你可以让玻璃幻灯片在PBS中坐几个小时,直到你准备好了。
  13. 从coplin罐中取出载玻片,让其风干5分钟。
  14. 通过在潮湿的塑料盒中应用100微升的封闭溶液1小时,阻止玻璃载玻片上的细胞。
  15. 通过将载玻片浸入装有PBS的Coplin罐中5分钟来洗涤细胞两次 注意:您不需要在洗涤期间摇动幻灯片或瓶子。
  16. 从coplin罐中取出载玻片,让其风干5分钟。
  17. 以1:500的稀释率加入50μl含一抗的PBS-T,并在潮湿的塑料盒中孵育细胞1小时。
  18. 通过将载玻片浸入装有PBS-T的coplin罐中5分钟来洗涤细胞六次
  19. 应用50微升含有PBS-T的二次抗体,稀释率为1:500,并在潮湿的塑料盒中孵育细胞1小时。保持在黑暗中。
  20. 通过将载玻片浸入装有PBS-T的coplin罐中5分钟来洗涤细胞六次
  21. 应用15微升安装溶液到细胞。确保没有气泡形成。
  22. 细胞现在准备通过荧光显微镜用特定的滤光片组(激发带通480/40和发射带通527/30)或具有488nm激光线的激光扫描共聚焦显微镜观察。载玻片可在4℃下在黑暗中储存一周。


    图2.间接免疫荧光测定的实际细胞图像和荧光信号 A.在步骤5后在载玻片上固定细胞。比例尺,10μm。 B.在步骤10之后在载玻片上固定细胞。提取叶绿素颜料并且细胞的颜色变透明。比例尺,10μm。 C.衣藻细胞的差异干涉对比图像。箭头指示其中富含CO 2 - 固定酶Rubisco的类固醇结构。比例尺,5μm。 D.源自抗LCIB-抗体的间接免疫荧光信号。 LCIB被检测为如先前报告中所示的类芘周围的环状结构(Yamano等人,2010)。比例尺,5μm。

笔记

我们强烈建议用户也执行相同的实验,使用目标基因突变体作为阴性对照,以区分真实信号和伪像。

食谱

  1. 4%甲醛
    1. 在搅拌板上将80ml PBS加热至约60℃。
    2. 加入4g多聚甲醛,并通过加入1N NaOH缓慢升高pH直至溶液变澄清。
    3. 溶解后,冷却并过滤溶液。
    4. 用PBS填充体积至100ml,用稀HCl调节pH至约6.9。
    5. 等分溶液可以在-20℃保存一个月。
  2. PBS
    将8g NaCl,0.2g KCl,1.44g Na 2 HPO 4和0.24g KH 2 PO 4溶解于水中, 4在900ml蒸馏的H 2 O中。用HCl调节pH至7.4,并将H 2 O加至1L。
  3. PBS-T
    向PBS中加入0.1%Tween-20(v/v)。
  4. 封锁解决方案
    PBS缓冲液中的5%(w/v)无球蛋白BSA

致谢

我们感谢王连勇的技术援助。这项工作得到了日本科学促进会科学奖25120714(H.F.)和25840109(T.Y.)和日本科学技术局先进低碳技术研究和发展计划的支持。

参考文献

  1. O anthon,N.,Mukherjee,B.,Tsujikawa,T.,Yanase,M.,Nakano,H.,Moroney,JV和Fukuzawa,H。(2010)。 
  2. Shakes,D.C.,Miller,D.M.,3rd and Nonet,M.L。(2012)。 免疫荧光显微镜。 Methods Cell Biol 107:35-66。
  3. Yamano,T.,Tsujikawa,T.,Hatano,K.,Ozawa,S.,Takahashi,Y.and Fukuzawa,H。(2010)。  合成碳酸氢盐摄入在绿藻中的叶绿体基质中的表征。 Proc Natl Acad Sci USA 112(23):7315-7320。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yamano, T. and Fukuzawa, H. (2016). Indirect Immunofluorescence Assay in Chlamydomonas reinhardtii. Bio-protocol 6(13): e1864. DOI: 10.21769/BioProtoc.1864.
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