A Simple Protocol for the Immunolabelling of Arabidopsis Pollen Tube Membranes and Cell Wall Polymers

引用 收藏 提问与回复 分享您的反馈 Cited by



Annals of Botany
Oct 2014



The pollen tube, a fast tip-growing cell, is an excellent model to study membrane and cell wall biosynthesis. Here, we describe a simple protocol using an easy to use device to perform immunofluorescence labelling of pollen tube membrane and cell wall. The use of the NucleoSpin column to perform all the steps of the immunolabelling procedure results in obtaining more intact pollen tubes.

Keywords: Pollen tube (花粉管), Cell wall (细胞壁), Membrane (膜), Immunolabelling (免疫标记)

Materials and Reagents

  1. Arabidopsis day-0 flowers according to Boavida and McCormick (2007)
    Note: Choose the last inflorescence buds opened at the top of the inflorescence stem. The stamen must not be higher than the stigma.
  2. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F0804 )
  3. Goat serum (Sigma-Aldrich, catalog number: G9023 )
  4. Saponin (Sigma-Aldrich, catalog number: S4521 )
  5. Primary antibody against plant cell wall polysaccharides/glycoproteins (http://www.plantprobes.net or http://www.ccrc.uga.edu/~carbosource) or digalactosyldiacylglycerol (DGDG) prepared in the laboratory using purified DGDG as antigen
  6. Secondary antibodies conjugated to Alexa Fluor 488 (Life Technology, catalog number: A-11008 ) or Fluorescein isothiocyanate (FITC) (Sigma-Aldrich, catalog number: F0382 )
  7. Arabidopsis pollen germination medium (PGM) (see Recipes)
  8. 1x Tris-buffered saline (TBS) (see Recipes)
  9. Fixation solution (see Recipes)
  10. Calcium- and magnesium-free Dulbecco's phosphate-buffered saline (CMF-DPBS) (see Recipes)
  11. Blocking buffer 1 (see Recipes)
  12. Blocking buffer 2 (see Recipes)


  1. Pair of tweezers
  2. Olympus CK2 inverted microscope
  3. Microscope Zeiss AxiObserver Z1
  4. Syringe Filter PVDF 33 mm 0.2 µm
  5. Microcentrifuge (VWRTM Galaxy 14D)
  6. NucleoSpin® Plasmid Binding Columns (Macherey-Nagel, catalog number: 740588.50 ), but columns of any other kits (Genejet or PureLink) may be used.
  7. Incubators at 22 °C and 30 °C
  8. Parafilm®
  9. Confocal Microscope TCS-SP2 operating system (Leica) and microscope Leica DMI6000B with a DFC450 C camera with FITC filter (absorption, 485-520 nm; emission, 520-560 nm wavelengths


  1. In vitro Arabidopsis pollen tube growth and fixation
    Note: See also the protocol available at Li (2011). However we advice to use the conditions detailed in the following protocol (number of flowers, PGM volume, temperature) to enhance the reproducibility.
    1. Grow Arabidopsis pollen in vitro according to the method described by Boavida and McCormick (2007).
      1. Harvest 40 freshly open Arabidopsis thaliana flowers (morning is the best) using a pair of tweezers.
      2. Submerge in 1 ml of PGM in a 1.5 ml Eppendorf tube.
      3. Shake the tube vigorously manually and with a vortex for 5 min to release the pollen grains from the anthers.
      4. Check the pollen number density with an inverted microscope.
      5. Remove all the flower debris with a pair of tweezers.
      6. Spin-down the pollen suspension using the microcentrifuge at 3,200 x g for 7 min (a yellowish pellet should be visible).
      7. Remove the supernatant by pipetting.
      8. Add 250 µl of PGM and resuspend the pellet.
      9. Place the tube horizontally in the incubator at 22 °C in the dark for 6 h.
      10. After 6 h, check for pollen germination and pollen tube growth with the inverted microscope (Figure 1).

        Figure 1. 6 h-old Arabidopsis pollen tubes. Note that the pollen grain density is important to obtain good pollen germination rates.

  2. Pollen tube fixation and immunolabelling
    Note: The protocol described below is adapted from Botté et al. (2011) and Dumont et al. (2014) for pollen membrane and pollen cell wall labelling respectively. Medium can be removed either by aspiration by connecting the NucleoSpin® Plasmid Binding Column to a vacuum such as a water aspirator (Figure 2) or by centrifugation at 300 x g for 1 min in a microcentrifuge. In the aspiration method, the flow-through is discarded and the germinated pollens present on the silica membrane are gently resuspended in the appropriate buffer. Although very similar the two protocols differ with an additional permeabilization step for pollen tube membrane labelling (step B4) and the use of different buffers (Figure 4). Buffers used for the cell wall labelling (Dumont et al., 2014) are written between parentheses.

    Figure 2. NucleoSpin® Plasmid Binding Columns connected to a hose for aspiration

    1. Pollen tubes in PGM were mixed (v/v) with a fixation medium containing 5% methanol-free formaldehyde in TBS buffer (or 5% formaldehyde in the fixation solution). Pollen tubes are incubated overnight at 4 °C before use (do not store the pollen tubes longer than 1 month).
    2. Transfer the pollen tubes solution on a NucleoSpin® Plasmid Binding Column held on a collection tube, under the fumehood.
      Note: All transfer and all washing steps described below are performed with a tip cut 0.5 cm from its extremity to avoid pollen tube damages (such ready to use wide bore pipet tips are available here: http://www.thomassci.com/Supplies/Pipettor-Tips/_/200l-Clear-Wide-Bore-Pipet-Tips?q=Wide%20Tip%20Pipet or http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=T-205-WB-C%28Lifesciences%29&categoryname=).
    3. Remove the medium by aspiration or centrifugation and wash three times with 100 µl TBS buffer (or CMF-DPBS, both filtered at 0.22 µm using a syringe filter). Pollen tubes are gently resuspended in the buffer by pipetting up and down and the buffer is removed by aspiration/centrifugation.
    4. Seal the bottom of the column with parafilm® and add 100 µl 0.002% saponin in TBS for 15 min for pollen membrane permeabilization (skip this step and the next one for the cell wall immunolabelling).
    5. Remove the medium by aspiration/centrifugation.
    6. Seal the bottom of the column with parafilm® and incubate the pollen tubes with 100 µl blocking buffer 1 (or blocking buffer 2), freshly prepared, for 1 h.
    7. Remove the blocking buffer by aspiration/centrifugation.
    8. The cell wall labelling requires three washes with CMF-DPBS as explained in step B3 (Skip this step for the membrane immunolabelling).
    9. Seal the bottom of the column and add 50 µl of primary antibody 1:10 dilution DGDG-specific rabbit serum in TBS-1%FBS-0.002% saponin and incubate 1 h at room temperature (or for the cell wall labelling: 1:20 primary antibody in CMF-DPBS overnight at 4 °C). Negative controls are prepared by omitting the primary antibody.
    10. Wash three times with TBS-1% FBS-0.002% saponin (or CMF-DPBS) as explained in step B3.
    11. Seal the bottom of the column and add 50 µl 1:500 dilution Alexa secondary antibody in TBS-1%FBS-0.002% saponin and incubate 1 h at room temperature (or for the cell wall labelling: FITC-coupled secondary antibody 1:50 dilution in CMF-DPBS for 2 h at 30 °C).
      Note: From this step, keep the tubes in the dark in aluminum foil.
    12. Wash four times in TBS-1% FBS-0.002% saponin and a final wash with TBS (or five times with CMF-DPBS) as explained in step B3.
    13. In order to recover the pollen tubes, resuspend them by adding at the top of the column 100 µl of TBS (or water) and by pipetting up and down several times with an enlarged tip and transfer the solution which contains the pollen tubes on a microscope slide with a cover slip. Figure 3 shows the confocal images showing the results of immunofluorescence labelling of Arabidopsis pollen tubes with anti-DGDG. Figure 4 gives an overview of the method for the membrane or cell wall immunolabelling of pollen tubes.

      Figure 3. Arabidopsis pollen grains were germinated for 6 h, pollen tubes were fixed and the pollen tube membrane immunolabelled with anti-DGDG antibodies. Alexa 488 was excited using the 488 nm line of an argon laser and fluorescence was collected between 500-539 nm. Superposition of control red autofluorescence induced by excitation at 543 nm laser is also shown.

      Figure 4. Schematic representation of the sample preparation for Arabidopsis pollen tubes plasma membrane and cell wall immunolabelling. Green dashed box indicates special step or buffer for plasma membrane labelling whereas blue dashed box points out the steps for the cell wall labelling. The picture at the bottom of the image shows a pollen tube labelled with an anti-rhamnogalacturonan-II (a pectic motif) antibody developed by Matoh et al. (1998) using a microscope Leica DMI6000B equipped with a DFC450 C camera (FITC filter: absorption, 485-520 nm; emission, 520-560 nm wavelengths).


  1. Arabidopsis Pollen Germination Medium (PGM)
    5 mM CaCl2
    0.01% H3BO3
    1 mM MgSO4
    5 mM KCl
    10% (w/v) sucrose
    pH 7.5
  2. 1x Tris-buffered saline (TBS)
    50 mM Tris-HCl
    150 mM NaCl
    pH 7.4
  3. Fixation solution
    100 mM PIPES buffer pH 6.9
    4 mM MgSO4 7H2O
    4 mM EGTA
    10% (w/v) sucrose
    5% (v/v) paraformaldehyde
  4. Calcium- and magnesium-free Dulbecco's phosphate-buffered saline (CMF-DPBS)
    137 mM NaCl
    2.7 mM KCl
    7 mM Na2HPO4 7H2O
    1.5 mM KH2PO4
    pH 7.2
  5. Blocking buffer 1
    1x TBS
    5% (w/v) FBS
    5% (w/v) goat serum
    0.002% Saponin
  6. Blocking buffer 2
    137 mM NaCl
    2.7 mM KCl
    7 mM Na2HPO4.7H2O
    1.5 mM KH2PO4
    pH 7.2
    3% fat-free milk
    Note: It is better to filter at 0.22 µm using a syringe filter all the washing solutions.


Original versions of the protocols were described in Dardelle et al. (2010), Botté et al. (2011) and Dumont et al. (2014). This work was supported by the University of Rouen, the region Haute Normandie, and the “Trans Channel Wallnet” project that was selected by the INTERREG IVA program France (Channel) – England European cross-border cooperation program, which is co-financed by the European Regional Development Funds (ERDF). The work performed at LPCV (Grenoble) was supported by the Agence Nationale de la Recherche (ANR-10-BLAN-1524, ReGal; ANR BioAdapt Reglisse).


  1. Boavida, L.C. and McCormick, S. (2007). TECHNICAL ADVANCE: Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J 52(3): 570-582.
  2. Botté, C.Y., Deligny, M., Roccia, A., Bonneau, A.L., Saïdani, N., Hardré, H., Aci, S., Yamaryo-Botté, Y., Jouhet, J., Dubots, E., Loizeau, K., Bastien, O., Bréhélin, L., Joyard, J., Cintrat, J. C., Falconet, D., Block, M. A., Rousseau, B., Lopez, R., and Maréchal, E. (2011). Chemical inhibitors of monogalactosyldiacylglycerol synthases in Arabidopsis thaliana. Nat Chem Biol 7(11): 834-842.
  3. Dardelle, F., Lehner, A., Ramdani, Y., Bardor, M., Lerouge, P., Driouich, A. and Mollet, J.C. (2010). Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall. Plant Physiol 153(4): 1563-1576.
  4. Dumont, M., Lehner, A., Bouton, S., Kiefer-Meyer, M. vC., Voxeur, A., Pelloux, J., Lerouge, P., and Mollet, J.C. (2014). The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein. Ann Bot 114(6): 1177-1188.
  5. PlantProbes: http://www.plantprobes.net
  6. CarboSource Services: http://www.ccrc.uga.edu/~carbosource
  7. Li, X. (2011). Arabidopsis pollen tube germination. Bio-protocol Bio101: e73.
  8. Matoh, T., Takasaki, M., Takabe, K., and Kobayashi, M. (1998) Immunocytochemistry of rhamnogalacturonan II in cell walls of higher plants. Plant Cell Physiol 39: 483–491.


花粉管,一种快速尖端生长细胞,是研究膜和细胞壁生物合成的优秀模型。 在这里,我们描述了一个简单的协议使用易于使用的设备进行免疫荧光标记花粉管膜和细胞壁。 使用NucleoSpin柱进行免疫标记过程的所有步骤导致获得更完整的花粉管。

关键字:花粉管, 细胞壁, 膜, 免疫标记


  1. 根据Boavida和McCormick(2007)的拟南芥 day-0花
    注意:选择在花序茎顶部打开的最后一个花序芽。 雄蕊不得高于柱头。
  2. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F0804)
  3. 山羊血清(Sigma-Aldrich,目录号:G9023)
  4. 皂苷(Sigma-Aldrich,目录号:S4521)
  5. 针对植物细胞壁多糖/糖蛋白的初级抗体( http://www.plantprobes.net http://www.ccrc.uga.edu/~carbosource )或双半乳糖基二酰基甘油(DGDG)在实验室中使用纯化的DGDG 作为抗原
  6. 缀合到Alexa Fluor 488(Life Technology,目录号:A-11008)或异硫氰酸荧光素(FITC)(Sigma-Aldrich,目录号:F0382)的二级抗体
  7. 拟南芥花粉萌发培养基(PGM)(参见Recipes)
  8. 1×Tris缓冲盐水(TBS)(见Recipes)
  9. 固定解决方案(参见配方)
  10. 无钙和镁的Dulbecco磷酸盐缓冲盐水(CMF-DPBS)(参见配方)
  11. 阻止缓冲区1(请参阅配方)
  12. 阻止缓冲区2(参见配方)


  1. 双镊子
  2. Olympus CK2倒置显微镜
  3. 显微镜Zeiss AxiObserver Z1
  4. 注射过滤器PVDF 33mm0.2μm
  5. 微量离心机(VWR TM Galaxy 14D)
  6. NucleosSpin质粒结合柱(Macherey-Nagel,目录号:740588.50),但可以使用任何其它试剂盒(Genejet或PureLink)的柱。
  7. 22℃和30℃的培养箱
  8. Parafilm ®
  9. 共聚焦显微镜TCS-SP2操作系统(Leica)和显微镜Leica DMI6000B与具有FITC滤光器的DFC450C照相机(吸收,485-520nm;发射,520-560nm波长


  1. 体外 拟南芥花粉管生长和固定
    注意:另见Li(2011)提供的协议。 但我们建议使用以下方案中详述的条件(花数,PGM体积,温度),以提高重现性。
    1. 根据Boavida和McCormick(2007)描述的方法,在体外生长拟南芥花粉 。
      1. 使用一对镊子收获40新鲜开放的拟南芥花(早上是最好的)。
      2. 浸入1ml PGM在1.5ml Eppendorf管中。
      3. 大力手动和用涡旋摇动管5分钟,以从花药释放花粉粒。
      4. 用倒置显微镜检查花粉数密度。
      5. 用一对镊子去除所有的花碎屑。
      6. 使用微量离心机在3,200×g下旋转花粉悬浮液7分钟(微黄色颗粒应该是可见的)。
      7. 通过吸移除去上清液。
      8. 加入250微升PGM并重悬沉淀
      9. 将管水平放置在孵化器中在22°C在黑暗中6小时。
      10. 6小时后,用倒置显微镜检查花粉萌发和花粉管生长(图1)。

        图1. 6 h-old拟南芥花粉管。请注意,花粉粒 密度对于获得良好的花粉萌发率是重要的。

  2. 花粉管固定和免疫标记
    注意:下面描述的协议改编自Bottéet al。 (2011)和Dumont et al。 (2014)分别为花粉膜和花粉细胞壁标记。可以通过将NucleoSpin 质粒结合柱连接到真空如吸水器(图2)在微量离心机中以300×g离心1分钟。在抽吸法中,弃去流出液,将存在于二氧化硅膜上的发芽的花粉轻轻地再悬浮于适当的缓冲液中。尽管非常相似,两个方案不同,另外的透化步骤用于花粉管膜标记(步骤B4)和使用不同的缓冲液(图4)。用于细胞壁标记的缓冲液(Dumont等人,2014)写在括号之间。

    图2. NucleoSpin ® 连接到软管的质粒结合柱

    1. 将PGM中的花粉管与含有固定介质的(v/v)混合 5%甲醇的TBS缓冲液(或5% 固定溶液)。将花粉管在4℃下孵育过夜 使用(不要存储花粉管超过1个月)。
    2. 将花粉管溶液转移到保持在收集管上的通风管下的NucleoSpin 质粒结合柱上。
      注意:执行以下所述的所有传送和所有清洗步骤 其尖端距离其末端0.5厘米,以避免花粉管损伤 (这样即可使用大口径移液器吸头在这里: http://www.thomassci.com/Supplies/Pipettor-Tips/_/200l-Clear-Wide-Bore-Pipet-Tips?q=Wide%20Tip%20Pipet http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=T-205-WB-C%28Lifesciences%29&categoryname= )。
    3. 通过抽吸或离心和洗涤去除介质 用100μlTBS缓冲液(或CMF-DPBS,两者均在0.22下过滤)洗涤三次 μm。 将花粉管轻轻地重悬在中 缓冲液通过上下吹吸,缓冲液被去除 抽吸/离心。
    4. 用柱子密封柱底部 parafilm ,并在TBS中加入100μl0.002%皂苷15分钟用于花粉 膜渗透(跳过此步骤,下一个用于细胞 壁免疫标记)
    5. 通过抽吸/离心去除培养基
    6. 用parafilm 密封柱的底部并孵育花粉   管中新鲜地加入100μl封闭缓冲液1(或封闭缓冲液2) 制备,1小时。
    7. 通过抽吸/离心去除阻塞缓冲液。
    8. 细胞壁标记需要用CMF-DPBS洗涤三次 解释在步骤B3(跳过此步骤为膜免疫标记)。
    9. 密封柱底部,加入50μl一级抗体1:10   稀释DGDG特异性兔血清在TBS-1%FBS-0.002%皂苷和 在室温孵育1小时(或细胞壁标记:1:20 一抗在CMF-DPBS中4℃过夜)。 阴性对照是 通过省略初级抗体制备
    10. 如步骤B3中所述用TBS-1%FBS-0.002%皂苷(或CMF-DPBS)洗涤三次。
    11. 密封柱的底部,加入50μl1:500稀释的Alexa 二抗在TBS-1%FBS-0.002%皂苷中,室温孵育1小时 温度(或对于细胞壁标记:FITC偶联的 抗体在CMF-DPBS中1:50稀释在30℃下2小时)。
    12. 在TBS-1%FBS-0.002%皂苷中洗涤四次,最后一次洗涤 TBS(或CMF-DPBS的五次),如步骤B3中所解释
    13. 在 为了恢复花粉管,通过在顶部添加重悬它们 的柱100μl的TBS(或水),并通过上下吹吸 几次用放大的尖端和转移的解决方案 包含具有盖玻片的显微镜载玻片上的花粉管。 图3显示了显示结果的共焦图像 用抗DGDG对拟南芥花粉管的免疫荧光标记。   图4给出了膜或细胞壁的方法的概述 花粉管的免疫标记

      图3. 拟南芥花粉颗粒  发芽6小时,花粉管固定,花粉管 膜与抗DGDG抗体免疫标记。 Alexa 488兴奋  使用488nm线的氩激光并收集荧光 在500-539nm之间。对照红色自发荧光的叠加 还示出了由在543nm激光下的激发诱导的

      图4。 拟南芥花粉管质膜和细胞壁免疫标记的样品制备的示意图。绿色虚线  框表示用于质膜标记的特殊步骤或缓冲液 而蓝色虚线框指出细胞壁的步骤 标签。图像底部的图片显示花粉管 用抗鼠李半乳糖醛酸聚糖-II(果胶基序)抗体标记 由Matoh等人(1998)使用显微镜Leica DMI6000B开发 装备有DFC450C相机(FITC过滤器:吸收,485-520nm; 发射,520-560nm波长)。


  1. 拟南芥花粉萌发培养基(PGM)
    5mM CaCl 2
    0.01%H 3 BO 3 sub。 1mM MgSO 4 5 mM KCl
    10%(w/v)蔗糖 pH 7.5
  2. 1×Tris缓冲盐水(TBS)
    50mM Tris-HCl
    150mM NaCl pH 7.4
  3. 固定溶液
    100mM PIPES缓冲液pH6.9
    4mM MgSO 4 7H 2 O 2·h/v 4mM EGTA
    10%(w/v)蔗糖 5%(v/v)多聚甲醛
  4. 无钙和镁的杜尔贝科磷酸盐缓冲盐水(CMF-DPBS)
    137 mM NaCl 2.7 mM KCl
    7mM Na 2 HPO 4 7H 2 O 2 1.5mM KH 2 PO 4 4/v/v pH 7.2
  5. 阻塞缓冲区1
    1x TBS
    5%(w/v)山羊血清 0.002%皂苷
  6. 阻塞缓冲区2
    137 mM NaCl 2.7 mM KCl
    7mM Na 2 HPO 4 sub 7H 2 O 1.5mM KH 2 PO 4 4/v/v pH 7.2


方案的原始版本描述于Dardelle等人(2010),Botté等人, (2011)和Dumont 等人(2014)。这项工作得到鲁昂大学,区域Haute Normandie和"INTER ChannelEGETA"项目选择的"Trans Channel Wallnet"项目的支持,该项目由法国(频道) - 英国欧洲跨境合作计划选定,该计划由联合资助欧洲区域发展基金(ERDF)。在LPCV(Grenoble)进行的工作由Agence Nationale de la Recherche(ANR-10-BLAN-1524,ReGal; ANR BioAdapt Reglisse)支持。


  1. 博瓦维达和McCormick,S。(2007)。 技术进步:温度作为增加和可再现性的决定性因素< (3):570-582。
  2. Botté,CY,Deligny,M.,Roccia,A.,Bonneau,AL,Saïdani,N.,Hardré,H.,Aci,S.,Yamaryo-Botté,Y.,Jouhet,J.,Dubots, Loizeau,K.,Bastien,O.,Bréhélin,L.,Joyard,J.,Cintrat,JC,Falconet,D.,Block,MA,Rousseau,B.,Lopez,R。,和Maréchal, )。 拟南芥中单半乳糖基二酰基甘油合成酶的化学抑制剂 < Nat Chem Biol 7(11):834-842
  3. Dardelle,F.,Lehner,A.,Ramdani,Y.,Bardor,M.,Lerouge,P.,Driouich,A.and Mollet,J.C。(2010)。 拟南芥花粉管细胞壁的生化和免疫细胞表征。 Plant Physiol 153(4):1563-1576。
  4. Dumont,M.,Lehner,A.,Bouton,S.,Kiefer-Meyer,M.v.C。,Voxeur,A.,Pelloux,J.,Lerouge,P.,and Mollet,J.C。(2014)。 细胞壁果胶聚合物rhamnogalacturonan-II是正常花粉管延长所必需的:推定的唾液酸转移酶like protein。 Ann Bot 114(6):1177-1188。
  5. PlantProbes: http://www.plantprobes.net
  6. CarboSource服务: http://www.ccrc.uga.edu/~carbosource
  7. Li,X.(2011)。 拟南芥 花粉管萌发 生物协议 Bio101:e73。
  8. Matoh,T.,Takasaki,M.,Takabe,K.,and Kobayashi,M.(1998)高等植物细胞壁中鼠李聚糖半乳糖醛酸II的免疫细胞化学。 植物细胞生理学39:483-491。
  • 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。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dumont, M., Cataye, C., Lehner, A., Maréchal, E., Lerouge, P., Falconet, D. and Mollet, J. (2015). A Simple Protocol for the Immunolabelling of Arabidopsis Pollen Tube Membranes and Cell Wall Polymers. Bio-protocol 5(12): e1502. DOI: 10.21769/BioProtoc.1502.