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Visualization of iron (Fe) localization in plants has greatly enhanced our understanding of plant Fe homeostasis. One of the relatively simple and yet powerful techniques is the classical Perls blue stain (Perls, 1867). The technique is based on the conversion of ferrocyanide to insoluble crystals of Prussian blue in the presence of Fe3+ under acidic conditions. It has been extensively used in animal and human histology (Meguro et al., 2007) and has recently gained popularity in plant research. For specific purposes, Fe signals may be additionally enhanced in the 3,3’-diaminobenzidine tetrahydrochloride (DAB) intensification procedure (Meguro et al., 2007). It has been demonstrated that this intensification results in the detection of both Fe2+ and Fe3+ ions (Roschzttardtz et al., 2009). The method has been successfully applied at the whole plant, organ and subcellular levels, both with (Roschzttardtz et al., 2011; Schuler et al., 2012; Roschzttardtz et al., 2013; Ivanov et al., 2014) and without intensification (Stacey et al., 2008; Long et al., 2010).
Here, we present a full Perls staining and DAB intensification protocol, the way it is performed in our lab (Ivanov et al., 2014).

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Perls Staining for Histochemical Detection of Iron in Plant Samples
普鲁士蓝染色用于植物样品中铁的组织化学检测

植物科学 > 植物细胞生物学 > 组织分析
作者: Tzvetina Brumbarova
Tzvetina BrumbarovaAffiliation: Institute of Botany, Heinrich-Heine University, Düsseldorf, Germany
Bio-protocol author page: a1670
 and Rumen Ivanov
Rumen IvanovAffiliation: Institute of Botany, Heinrich-Heine University, Düsseldorf, Germany
For correspondence: rumen.ivanov@uni-duesseldorf.de
Bio-protocol author page: a1671
Vol 4, Iss 18, 9/20/2014, 5482 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1245

[Abstract] Visualization of iron (Fe) localization in plants has greatly enhanced our understanding of plant Fe homeostasis. One of the relatively simple and yet powerful techniques is the classical Perls blue stain (Perls, 1867). The technique is based on the conversion of ferrocyanide to insoluble crystals of Prussian blue in the presence of Fe3+ under acidic conditions. It has been extensively used in animal and human histology (Meguro et al., 2007) and has recently gained popularity in plant research. For specific purposes, Fe signals may be additionally enhanced in the 3,3’-diaminobenzidine tetrahydrochloride (DAB) intensification procedure (Meguro et al., 2007). It has been demonstrated that this intensification results in the detection of both Fe2+ and Fe3+ ions (Roschzttardtz et al., 2009). The method has been successfully applied at the whole plant, organ and subcellular levels, both with (Roschzttardtz et al., 2011; Schuler et al., 2012; Roschzttardtz et al., 2013; Ivanov et al., 2014) and without intensification (Stacey et al., 2008; Long et al., 2010).
Here, we present a full Perls staining and DAB intensification protocol, the way it is performed in our lab (Ivanov et al., 2014).
Keywords: Perls staining(Perls染色), DAB intensification(DAB的强化), Iron deficiency(铁缺乏), Plant(植物)

[Abstract]

Materials and Reagents

  1. 3,3’-diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich, catalog number: 32750 )
  2. Chloroform (CHCl3)
  3. Cobalt (II) chloride (CoCl2)
  4. Ethanol (CH3CH2OH)
  5. Glacial (water-free) acetic acid (CH3COOH)
  6. Hydrogen peroxide (H2O2) (30%)
  7. Hydrochloric acid (HCl) (37%)
  8. Methanol (CH3OH)
  9. Di-sodium hydrogenphosphate (Na2HPO4)
  10. Sodium di-hydrogenphosphate (NaH2PO4)
  11. Sodium azide (NaN3)
  12. Potassium ferrocyanide (K4[Fe (Cn)6])
  13. Fixing solution (see Recipes)
  14. Staining solution (see Recipes)
  15. 0.1 M phosphate buffer (see Recipes)
  16. 1% 3,3’-diaminobenzidine tetrahydrochloride (DAB) stock (see Recipes)
  17. Preparation solution (see Recipes)
  18. 1% CoCl2 (see Recipes)
  19. Intensification solution (see Recipes)

Equipment

  1. Vacuum pump (any model capable of producing 500 mbar vacuum)
  2. 1.5 ml tube
  3. Standard incubator

Procedure

  1. Perls stain
    1. Fix plant material for 1-2 h under vacuum (500 mbar) in the fixing solution. Usually this can be done with 1 ml solution in a standard 1.5 ml tube. Larger samples will require recalculating the volumes. Vacuum application allows rapid penetration of the fixative. Samples need to be completely submerged. Using of vacuum desiccator is fully sufficient for this step. During this time, prepare the staining solution and pre-warm it at 37 °C in a standard incubator.
    2. Remove the fixing solution.
    3. Wash 3 times for 1-2 min each with distilled water.
    4. Add the pre-warmed staining solution and incubate for 15 min to 1 h under vacuum (500 mbar). Again, the usual volume is 1 ml solution in a 1.5 ml tube.
    5. Remove the staining solution.
    6. Wash 3 times for 1-2 min each with distilled water. Gentle shaking might be applied.
    7. Store in distilled water. It is advisable to analyze, or image, the samples within one week.
    8. (Optional step) Dehydration: Incubate in an ethanol dilution series: 10%, 30%, 50%, 70%. Dehydration will intensify the blue stain. This step is not recommended if further intensification of the signal is planned. For samples with high iron content, such as roots or samples from metal hyperaccumulating plants, Perls stain alone may be fully sufficient for obtaining intensive signal. If the aim is a detailed view of tissue or cellular level iron detection the additional intensification should be performed (see below).

  2. DAB intensification
    1. Incubate the samples, prepared and washed in distilled water in step A6, for 1 h with the preparation solution. No vacuum is applied here.
    2. Remove the solution and wash three times with 0.1 M Phosphate buffer.
    3. Incubate the samples with Intensification solution at room temperature. Brown staining may appear as early as five min after the beginning of the incubation. In any case, the reaction should not continue for more than 30 min.
    4. To stop the reaction, remove the Intensification solution and wash three times with distilled water.
    5. Store samples in distilled water.

Notes

  1. Incubation of plants in Fe-containing agar medium (usually around 50 µM Fe) will result in strong staining of the root apoplast. If the aim of the experiment is to observe whole-mount Fe distribution under standard Fe supply in inner parts of the root, such as the central cylinder, plants should be grown on soil.

Representative Data



Figure 1. Visualization of Fe in Arabidopsis root by Perls staining and DAB intensification.
A. A fixed 7 day-old Arabidopsis seedling. B. Perls-stained root. Signal is seen in the apoplast and central cylinder. C. DAB intensification applied without prior Perls staining. No specific signal can be seen. D. Perls-DAB staining on a root, grown on agar plate under sufficient Fe supply. Strong signal can be seen in the apoplast. E. Perls-DAB staining on a root, grown on agar plate under sufficient Fe supply and then transferred for 24 to Fe-deficient medium. Staining can be seen in the central cylinder. Low to no signal is present at the apoplast. The zone represented on the closeup images corresponds to the highlighted zone in A. Bars represent 1 mm.

Recipes

  1. Fixing solution
    Methanol: Chloroform: Glacial acetic acid (6:3:1)
    The three can be pipetted in any order. The end solution should contain only one phase.
  2. Staining solution
    4% K4Fe(Cn)6 (4 g per 100 ml in distilled water) and 4% HCl (10.81 ml from a 37% HCl stock solution to a final volume of 100 ml) stock solutions are mixed in a 1:1 (v:v) proportion. It is advisable to store the two stock solutions in the dark at room temperature and prepare the mixture fresh before use.
  3. 0.1 M phosphate buffer
    Prepare 1 M stocks of Na2HPO4 and NaH2PO4. For 50 ml 0.1 M buffer at pH 7.0, mix 2.89 ml of the Na2HPO4 and 2.12 ml of the NaH2PO4. Add distilled water to a volume of 45 ml and measure the pH. If needed, adjust with sodium hydroxide (NaOH) or phosphoric acid (H3PO4). Then add distilled water to a final volume of 50 ml. pH value of the buffer is critical for the quality of the staining and may be varied, depending on the exact application. pH values under 7.0 will lead to reduction in staining intensity. pH over 7.6 may increase the background staining.
  4. 1% 3,3’-diaminobenzidine tetrahydrochloride (DAB) stock
    Several (10-15) microliters of 10 M (37%) HCl should be added per each milliliter of solution until the color becomes light brown. Shake for five minutes to help the complete dissolving of DAB. However, aggregates of undissolved DAB may remain. In this case, it is advisable to filter the solution through a 0.2 µm filter. We have not tried to dissolve the remaining clumps by sonication.
  5. Preparation solution
    0.01 M NaN3 and 0.3 % H2O2 in methanol
  6. 1% CoCl2
    Stock solution needed for the preparation of the intensification solution
    Can be stored for long periods at room temperature
  7. Intensification solution
    0.1 M Phosphate buffer (pH 7.0) containing 0.025% DAB, 0.005% H2O2 and 0.005% CoCl2
    Prepare fresh

Acknowledgements

This protocol is based on the procedure described by Roschzttardtz et al. (2009). Research in the authors’ laboratory was supported by the Saarland University, Germany, and the Heinrich Heine University, Germany. We would like to thank Ailisa Blum for helping introduce the technique in our lab.

References

  1. Ivanov, R., Brumbarova, T., Blum, A., Jantke, A.-M., Fink-Straube, C. and Bauer, P. (2014). SORTING NEXIN1 is required for modulating the trafficking and stability of the Arabidopsis IRON-REGULATED TRANSPORTER1. Plant Cell 26(3): 1294-1307.
  2. Long, T. A., Tsukagoshi, H., Busch, W., Lahner, B., Salt, D. E. and Benfey, P. N. (2010). The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots. Plant Cell 22(7): 2219-2236.
  3. Meguro, R., Asano, Y., Odagiri, S., Li, C., Iwatsuki, H. and Shoumura, K. (2007). Nonheme-iron histochemistry for light and electron microscopy: a historical, theoretical and technical review. Arch Histol Cytol 70(1): 1-19.
  4. Perls, M. (1867). Nachweis von Eisenoxyd in gewissen Pigmenten. Archiv für pathologische Anatomie und Physiologie und für klinische Medicin 39(1): 42-48.
  5. Roschzttardtz, H., Conéjéro, G., Curie, C. and Mari, S. (2009). Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo. Plant Physiol 151(3): 1329-1338.
  6. Roschzttardtz, H., Grillet, L., Isaure, M.-P., Conéjéro, G., Ortega, R., Curie, C. and Mari, S. (2011). Plant cell nucleolus as a hot spot for iron. J Biol Chem 286(32): 27863-27866.
  7. Roschzttardtz, H., Conéjéro, G., Divol, F., Alcon, C., Verdeil, J.-L., Curie, C. and Mari, S. (2013). New insights into Fe localization in plant tissues. Fron Plant Sci 4.
  8. Schuler, M., Rellan-Alvarez, R., Fink-Straube, C., Abadia, J. and Bauer, P. (2012). Nicotianamine functions in the phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis. Plant Cell 24(6): 2380-2400.
  9. Stacey, M. G., Patel, A., McClain, W. E., Mathieu, M., Remley, M., Rogers, E. E., Gassmann, W., Blevins, D. G. and Stacey, G. (2008). The Arabidopsis AtOPT3 protein functions in metal homeostasis and movement of iron to developing seeds. Plant Physiol 146(2): 589-601.

材料和试剂

  1. (DAB)(Sigma-Aldrich,目录号:32750)
  2. 氯仿(CHCl 3)
  3. 氯化钴(II)(CoCl 2)
  4. 乙醇(CH 3 CH 2 OH)
  5. 冰醋酸(无水)乙酸(CH 3 COOH)
  6. 过氧化氢(H 2 O 2 sub)(30%)
  7. 盐酸(HCl)(37%)
  8. 甲醇(CH 3 OH)
  9. 磷酸氢二钠(Na 2 HPO 4)
  10. 磷酸氢二钠(NaH 2 PO 4)
  11. 叠氮化钠(NaN 3)
  12. 亚铁氰化钾(K 4+ [Fe(C n)6])
  13. 固定解决方案(参见配方)
  14. 染色溶液(见配方)
  15. 0.1 M磷酸盐缓冲液(见配方)
  16. 1%3,3'-二氨基联苯胺四盐酸盐(DAB)原料(见配方)
  17. 准备解决方案(参见配方)
  18. 1%CoCl <2> (参见配方)
  19. 强化解决方案(参见配方)

设备

  1. 真空泵(任何能够产生500 mbar真空的型号)
  2. 1.5 ml管
  3. 标准孵化器

程序

  1. Perls染色
    1. 在固定溶液中在真空(500毫巴)下固定植物材料1-2小时。 通常这可以用标准1.5ml管中的1ml溶液进行。 较大的样品将需要重新计算体积。 真空应用允许固定剂的快速渗透。 样品需要完全淹没。 使用真空干燥器完全足以用于该步骤。 在此期间,准备染色溶液,并在标准培养箱中在37℃预热
    2. 取出固定溶液。
    3. 用蒸馏水洗涤3次,每次1-2分钟
    4. 加入预热的染色溶液,并在真空(500毫巴)下孵育15分钟至1小时。同样,通常的体积是在1.5ml管中的1ml溶液
    5. 取出染色溶液。
    6. 用蒸馏水洗涤3次,每次1-2分钟。可能会轻微摇动。
    7. 储存在蒸馏水中。建议在一周内分析或成像样品。
    8. (任选步骤)脱水:在乙醇稀释系列中孵育:10%,30%,50%,70%。脱水会加剧蓝色污渍。如果计划进一步强化信号,则不推荐这一步骤。对于具有高铁含量的样品,例如来自金属高度积累植物的根或样品,单独的Perls染色可能完全足以获得强信号。如果目的是详细查看组织或细胞水平铁检测,应进行额外的强化(见下文)
  2. DAB集约化
    1. 孵育样品,准备并在蒸馏水中洗涤步骤A6,用制备溶液1小时。 此处不施加真空。
    2. 取出溶液,用0.1M磷酸盐缓冲液洗涤三次
    3. 在室温下用强化溶液孵育样品。 棕色染色可以早在孵育开始后5分钟出现。 在任何情况下,反应不应持续超过30分钟。
    4. 要停止反应,取出增溶溶液,并用蒸馏水洗涤三次
    5. 将样品储存在蒸馏水中。

笔记

  1. 植物在含Fe琼脂培养基(通常约50μMFe)中的孵育将导致根原生质体的强染色。 如果实验的目的是观察在根部内部部分(例如中心圆柱)的标准Fe供应下的整体Fe分布,植物应当在土壤上生长。

代表数据



图1.通过Perls染色和DAB强化在拟南芥根中显现Fe。 A.固定的7日龄拟南芥幼苗。 B. Perls染色的根。 信号在质外体和中心柱体中看到。 C. DAB 在没有预先Perls染色的情况下施加强化。没有可以看到特定的信号。 D.在足够的Fe供应下在琼脂平板上生长的根上的Perls-DAB染色。强的信号可以在质外体中看到。 E. Perls-DAB在根上生长,在足够的Fe供应下在琼脂平板上生长,然后转移到Fe缺乏培养基中24小时。可以在中心圆筒中看到染色。在质外体处存在低至无信号。特写图像上表示的区域对应于A中的突出显示的区域。条表示1mm。

食谱

  1. 固定解决方案
    甲醇:氯仿:冰醋酸(6:3:1)
    三个可以任何顺序移液。最终解决方案应该只包含一个阶段。
  2. 染色溶液
    4%K 4 Fe(C n)6(4g/100ml,在蒸馏水中)和4%HCl(10.81ml,从37%HCl储备溶液至最终体积的100ml)储备溶液以1:1(v:v)比例混合。建议将两种储备溶液在室温下在黑暗中储存,并在使用前将混合物清洗干净
  3. 0.1 M磷酸盐缓冲液
    制备Na 2 HPO 4和NaH 2 PO 4的1M储备液。对于pH 7.0的50ml 0.1M缓冲液,混合2.89ml Na 2 HPO 4和2.12ml NaH 2 PO 4 > 4 。加入蒸馏水至体积为45ml,并测量pH。如果需要,用氢氧化钠(NaOH)或磷酸(H 3 PO 4)调节。然后加入蒸馏水至终体积为50ml。缓冲液的pH值对于染色的质量是关键的,并且可以根据确切的应用而变化。 pH值低于7.0将导致染色强度的降低。 pH高于7.6可增加背景染色。
  4. 1%3,3'-二氨基联苯胺四盐酸盐(DAB) 每毫升溶液应加入几(10-15)微升的10M(37%)HCl,直至颜色变为浅棕色。摇动5分钟以帮助DAB完全溶解。然而,可能保留未溶解的DAB的聚集体。在这种情况下,建议通过0.2μm过滤器过滤溶液。我们还没有试图通过超声处理来溶解剩余的团块
  5. 制备溶液
    0.01M NaN 3和0.3%H 2 O 2在甲醇中的溶液。
  6. 1%CoCl 2
    制备强化溶液所需的储备溶液
    可在室温下长期保存
  7. 增强解决方案
    含有0.025%DAB,0.005%H 2 O 2和0.005%CoCl 2的0.1M磷酸盐缓冲液(pH 7.0) 准备新鲜

致谢

该方案基于Roschzttardtz等人描述的方法。 (2009)。 作者实验室的研究得到了德国萨尔州大学和德国海因里希·海涅大学的支持。 我们要感谢Ailisa Blum帮助在我们的实验室中介绍这项技术。

参考文献

  1. Ivanov,R.,Brumbarova,T.,Blum,A.,Jantke,A.-M.,Fink-Straube,C.and Bauer,P。 SORTING NEXIN1是调整 植物细胞26(3):1294-1307。植物细胞的转运和稳定性拟南芥 IRON-REGULATED TRANSPORTER1。
  2. Long,T.A.,Tsukagoshi,H.,Busch,W.,Lahner,B.,Salt,D.E。和Benfey,P.N。(2010)。 bHLH转录因子POPEYE调节拟南芥根中对铁缺乏的反应。 植物细胞 22(7):2219-2236
  3. Meguro,R.,Asano,Y.,Odagiri,S.,Li,C.,Iwatsuki,H.and Shoumura,K。(2007)。 gewissen Pigmenten中的Nachweis von Eisenoxyd。 Archivfürpathologische Anatomie und Physiologie undfürklinische Medicin 39(1):42-48。
  4. Roschzttardtz,H.,Conéjéro,G.,Curie,C.and Mari,S。(2009)。 鉴定拟南芥中的内胚层液泡作为铁储存室 >胚胎 植物生理 151(3):1329-1338
  5. Roschzttardtz,H.,Grillet,L.,Isaure,M.-P.,Conéjéro,G.,Ortega,R.,Curie,C.and Mari,S。(2011)。 植物细胞核仁作为铁的热点 J Biol Chem 286(32):27863-27866。
  6. Roschzttardtz,H.,Conéjéro,G.,Divol,F.,Alcon,C.,Verdeil,J.-L.,Curie,C.and Mari, 关于植物组织中Fe定位的新见解。 Fron Plant Sci 4.
  7. Schuler,M.,Rellan-Alvarez,R.,Fink-Straube,C.,Abadia,J.and Bauer,P.(2012)。 尼古丁胺在基于韧皮部的铁运输中吸收器官,在花粉发育和花粉管生长中起作用in Arabidopsis 。 Plant Cell 24(6):2380-2400。
  8. Stacey,M.G.,Patel,A.,McClain,W.E.,Mathieu,M.,Remley,M.,Rogers,E.E.,Gassmann,W.,Blevins,D.G.and Stacey,G。(2008)。 拟南芥AtOPT3蛋白在金属内稳态和铁向发育中的运动中的功能种子。植物生理学146(2):589-601。
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How to cite this protocol: Brumbarova, T. and Ivanov, R. (2014). Perls Staining for Histochemical Detection of Iron in Plant Samples. Bio-protocol 4(18): e1245. DOI: 10.21769/BioProtoc.1245; Full Text



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11/18/2014 7:15:07 AM  

Paula Pongrac
University of Bayreuth

Hi Rumen,

did you ever observe that parts of the tissue remains blue after intensification?

Thanks,
Paula

11/19/2014 12:33:20 AM  

Rumen Ivanov (Author)
Institute of Botany, Heinrich-Heine University, Germany

Dear Paula,

Recently, we had cases of blue staining remaining in the base of the root (next to the hypocotyl). This happened with plants that take up more iron and the overall iron content was strongly elevated.

I suppose the fixation and infiltration conditions might have to be optimized in this case but we have not tackled the problem yet. I will write back if we find a solution for this one.

Rumen

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10/1/2014 7:28:10 AM  

Paula Pongrac
University of Bayreuth

Hi,

I am wondering why the fixation is needed prior staining. I perform staining without fixation step and it works nicely, but I prefer to take photos immediately, as I saw some changes in intensities after one day. I would have a question: do you think potassium phosphate buffer would yield different results?

Thanks,

Paula

10/2/2014 5:27:39 AM  

Rumen Ivanov (Author)
Institute of Botany, Heinrich-Heine University, Germany

Dear Paula,

thanks for the information. We have never used unfixed material for the procedure. To my knowledge, the problem lies with DAB, since in living tissues it would react with the endogenously generated hydrogen peroxide. This might be a problem when the plant is heavily stressed in the steps preceding the DAB addition.

For the potassium phosphate buffer, we did not use that either but my guess is that it should work the same way the sodium buffer.

Best wishes,

Rumen

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