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Phalloidin Staining and Immunohistochemistry of Zebrafish Embryos
斑马鱼胚胎的鬼笔环肽(phalloindin)染色和免疫组织化学分析   

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Abstract

Fluorescent conjugated Phalloidin is a stain that allows for visualization of F-actin. In immunohistochemistry, primary antibodies and fluorescent conjugated secondary antibodies can be used to visualize subcellular localization and relative amounts of proteins of interest. Here is a protocol for Phalloidin and antibody staining of zebrafish embryos 5 days old and younger.

Keywords: Muscle(肌肉), Myotendinous junction(交接处), Antibody(抗体)

Materials and Reagents

  1. Alexa Fluor 488 or 546 Phalloidin (Life Technologies)
  2. Desired primary antibodies (see Table 1 for information for antibodies commonly used in the Henry Lab)
  3. Alexa Fluor 488, 546, or 633 secondary antibodies (e.g. goat anti-mouse or goat anti-rabbit secondary antibodies, Life Technologies)
  4. Vacuum grease (e.g. Dow Corning High vacuum grease)
  5. 10x PBS (see Recipes)
  6. PBS 0.1% Tween-20® (see Recipes)
  7. PBS 2% Tween-20® (see Recipes)
  8. 8% PFA (see Recipes)
  9. 4% PFA (see Recipes)
  10. Block (see Recipes)
  11. 80:20 glycerol:PBS solution (see Recipes)

Equipment

  1. Two fine forceps (e.g. Dumont #5 tweezers)
  2. Two deyolking tools (e.g. insect pin super glued in the end of a glass capillary tube, Figure 1)
  3. Bench rocker
  4. 1.5-2 ml Microcentrifuge tubes
  5. Glass Pasteur pipettes
  6. Pipette pump
  7. Micropipettes
  8. Micropipette tips
  9. Microscope slides
  10. Microscope slides
  11. Dissecting microscope
  12. Microscope for image acquisition (e.g. Zeiss Axio Imager running AxioVision software)


    Figure 1. Deyolking tools. Deyolking tools can be used to surgically remove the yolk sac from fixed zebrafish embryos. Deyolking tools consist of insect pins (Fine Science Tools, catalog number: 26002-20) super glued into the ends of glass capillary tubes (Sutter Instruments, catalog number: BF100-50-10). The glass capillary tubes are then wrapped in lab tape.

Procedure


A. Fixation and Phalloidin staining:

  1. Dechorionate embryos with two pairs of fine forceps. Under a dissecting microscope, pinch an embryo’s chorion using a pair of forceps held in one of your hands. With the forceps in your other hand, pinch the chorion near to the original pinch and gently tear the chorion by separating your hands. Repeat pinching and tearing chorions with forceps until the embryos are dechorionated.
  2. Using a pipette pump and glass pipettes, transfer dechorionated embryos into microcentrifuge tubes. Put a maximum of 10 embryos in a single tube. Label the tubes accordingly.
    Note: A pipette pump and glass pipette tips can be used for all solution additions and removals in this protocol excluding the addition of primary and secondary antibodies. Gently pipetting the embryos up and down in the glass pipette tip with each solution change improves washing and keeps the embryos from sticking together.
  3. Remove as much liquid as possible from the microcentrifuge tubes.
  4. Wear gloves when working with PFA. Add ~0.5 ml of 4% PFA to each microcentrifuge tube.
  5. Place the microcentrifuge tubes containing fixative on their sides at room temperature on a bench rocker (gently rocking) for 4 h or at 4 °C overnight. Orient the tubes perpendicular to the rocking motion such that the embryos rock from side to side in the tube rather than from cap to bottom.
  6. Remove the fixative and dispose of it in the appropriate waste container.
  7. Rinse the embryos 3 times for 5 min each in ~0.5 ml PBS 0.1% Tween-20®. With each rinse in this protocol, gently resuspend the embryos in the solution to improve rinsing and keep them from sticking together.
  8. Remove the last PBS 0.1% Tween-20® rinse and add ~0.5 ml PBS 2% TritonX-100® to each tube to permeabilize the embryos for Phalloidin staining.
  9. Lay the tubes containing PBS 2% Trition on their sides and gently rock for 1.5 h at room temperature.
  10. Remove the PBS 2% TritonX-100® and use a P20 micropipette to add 19 μl of PBS 2% TritonX-100® to each tube. Use a P20 micropipette to add 1 μl of Alexa Fluor 488 or 546 Phalloidin to each tube (wear gloves when working with Phalloidin).
  11. Lay the tubes on their sides and gently rock (if possible) overnight at 4 °C. From this point on, keep tubes in the dark whenever possible (e.g. under a box lid or wrapped in foil).


B. Antibody staining with mono/polyclonal antibodies:

  1. Remove Phalloidin from the tubes and add ~0.5 ml of block to each tube.
  2. Rock tubes containing block on their sides in the dark for at least 1 h at room temperature.
  3. Use micropipettes to add block and primary antibody solutions into each tube to obtain the appropriate primary antibody dilution optimized for zebrafish embryo staining. A 1:100 dilution of primary antibody in block is a good place to start. Primary antibody dilutions can range from 1:10 to 1:5,000. Some antibodies may require additional permeabilization steps (e.g. incubation in methanol or acetone at -20 °C, proteinase treatment) prior to the addition of the primary antibody. Some primary antibodies may not be compatible with Phalloidin staining.
  4. Incubate embryos in primary antibody at 4 °C overnight in the dark with tubes on their sides and gently rocking, if possible.
  5. Remove the primary antibody dilution and add in ~0.5 ml block.
  6. Block embryos with tubes on their sides in the dark and gently rocking for ~8 h at room temperature. Additional rinses with block can be added if necessary to reduce non-specific background staining.
  7. Remove block and use micropipettes to add 199 μl of antibody block and 1 μl of the appropriate fluorescent conjugated secondary antibody into each tube. Be mindful of the wavelength of the fluorophore that you used for Phalloidin staining when determining the appropriate secondary antibody to use. Ensure that the secondary antibody used corresponds to whether the primary antibody is monoclonal or polyclonal.
  8. Incubate embryos in this secondary antibody dilution overnight at 4 °C in the dark with tubes on their sides and gently rocking, if possible.
  9. Remove secondary antibody dilution and store embryos in ~0.5 ml PBS 0.1% Tween-20® in the dark. Additional rinses in PBS 0.1% Tween-20® can be added if necessary to reduce non-specific background staining.


C. Deyolking, Mounting, and Imaging:

  1. Manually deyolk embryos in 1x PBS with two deyolking tools. Use a deyolking tool to orient an embryo on its side. Hold the embryo down by inserting the tip of the deyolking tool held in one hand at a location in between the yolk sac and the embryo’s body. Use the deyolking tool in the other hand to ‘cut’ the yolk sac away from the embryo’s body while keeping the other hand stationary. Once the majority of yolk has been removed, use the pins as desired to clean up and remove any additional yolk sac remnants. Suck up and discard the pieces of yolk sac. Repeat deyolking process until all embryos are deyolked.
  2. Transfer embryos to 80:20 glycerol:PBS. If embryos become dehydrated upon transfer to 80:20 glycerol:PBS, slow washes in increasing concentrations of glycerol may have to be implemented. For example, 1 h long washes in each of 30:70 glycerol:PBS and 60:40 glycerol:PBS may be necessary before transfer to 80:20 glycerol:PBS. Use one deyolked embryo as a test sample and move it from 1x PBS to 80:20 glycerol:PBS. If the shape of the embryo changes, proceed with the incubations listed above for the rest of the embryos.
  3. Dot a glass microscope slide with 4 small dots of vacuum grease such that the dots will hold up the four corners of a square cover slip.
  4. Transfer one embryo and two drops of 80:20 glycerol:PBS onto the microscope slide in the middle of the 4 dots of grease.
  5. Orient the embryo with a deyolking tool so that it is side mounted and flat.
  6. Place the cover slip over the specimen and gently press down on the corners of the cover slip until it is touching the embryo, but not squishing it.
  7. Repeat steps C3-6 until all embryos are mounted. Keep your prepared slides in a slide book in the dark at 4 °C until they are imaged. Image your stained zebrafish embryos within ~2 weeks, the sooner the better. Representative images are shown in Figure 2.


    Figure 2. Phalloidin staining representative results. (A) Brightfield image of a deyolked, side mounted zebrafish embryo, anterior left, dorsal top. (B) Fluorescence micrograph of the same embryo showing phalloidin 546 staining. (C) Image of the same embryo taken with the 20x objective on a Zeiss Axio Imager running AxioVision software. Phalloidin 546 staining enables visualization of the actin cytoskeleton of skeletal muscle fibers. For representative images of antibody staining, please refer to (Goody et al., 2010).

    Table 1. Antibody staining information for antibodies commonly used in the Henry Lab.

    Name Company
    Product #
    Dilution
    Mono/polyclonal
    Works with phalloidin®
    Extra


    permeabiliza-tion®

    Beta-Dystroglycan
    Novocastra
    NCLbDG
    1:50
    Monoclonal
    Yes
    No
    F59
    Developmental Studies Hybridoma Bank (DSHB)

    1:10
    Monoclonal
    Yes
    No
    Fibronectin
    Sigma
    F3648
    1:50
    Polyclonal
    Yes
    No
    Laminin-111
    Thermo Scientific
    RB-082-A0
    1:50
    Polyclonal
    Yes
    No
    Paxillin
    BD Transduction Laboratories
    610051
    1:50
    Monoclonal
    Yes
    No
    FAK pY397 or pY861
    Invitrogen

    1:50
    Polyclonal
    Yes
    No
    Dystrophin
    Sigma
    D8043
    1:50
    Monoclonal
    Yes
    No
    MF20
    DSHB

    1:10
    Monoclonal
    Not well
    No
    F310
    DSHB

    1:10
    Monoclonal
    Not well
    No
    Vinculin
    Sigma
    V4505
    1:10
    Monoclonal
    Unknown
    PBS 2% TritonX-100® for 2.5 hours at room temp
    4D9/engrailed
    DSHB

    1:2
    Monoclonal
    Yes
    10 minutes in acetone at -20 °C
    Beta-catenin
    Abcam
    Ab6302
    1:500
    Monoclonal
    Unknown
    Requires special fixative (4% PFA, 4% sucrose, 3 mM CaCl2, 1x PBS)
    GFP
    Molecular Probes
    A21311
    1:200
    Polyclonal
    Unknown
    2 h fix in 4% PFA at room temp

Recipes

  1. Phosphate Buffered Saline (PBS)
    1. 10x PBS
      Add to a 1 L bottle
      74 g NaCl
      19.4 g Na2HPO4.7H2O
      4.37 g NaH2PO4.H2O
      ~800 ml dH2O
      Stir until dissolved. Bring volume up to 1 L with dH2O. Autoclave. Dilute 10x PBS to 2x PBS or 1x PBS with dH2O. Store at room temperature.
    2. PBS 0.1% Tween-20®
      To 1 L of 1x PBS, add
      1 ml of Tween-20®
      Mix solution. Store at room temperature.
    3. PBS 2% TritonX-100®
      To 1 L of 1x PBS, add
      20 ml TritonX-100®
      Mix solution. Store at room temperature.
  2. Paraformaldehyde (PFA)
    1. 8% PFA
      Add to a 50 ml conical tube
      4 g PFA
      30 ml dH2O
      20 drops 1 N NaOH
      Gently heat and stir until dissolved. Bring volume up to 50 ml with dH2O. Filter solution through #1 Whatman paper. Add 20 drops 1 N HCl and mix. Store at 4 °C. Use within 1 week.
    2. 4% PFA
      Add to a 50 ml conical tube
      25 ml 8% PFA
      25 ml 2x PBS
      Mix, then store at 4 °C. Use within 1 week.
  3. Block
    Add to a 50 ml conical tube-
    2.5 g Bovine Serum Albumin (BSA)
    40 ml 1x PBS
    Gently heat and stir until BSA is dissolved. Then add
    0.5 ml DMSO
    0.5 ml TritonX-100®
    0.1 g Saponin
    Bring up to 50 ml with 1x PBS. Store at 4 °C. Use within 1 week.
  4. 80:20 glycerol:PBS
    Add to a 50 ml conical tube-
    40 ml glycerol
    10 ml 1x PBS
    Mix solution. Store at room temperature

Acknowledgments

This protocol was adapted from the previous publications: Goody et al. (2010) and Goody et al. (2012). Development of this protocol was supported by NIH grant RO1 HD052934-01A1 to CAH. MFG would like to thank the University of Maine Graduate School of Biomedical Sciences and Engineering for funding.

References

  1. Goody, M. F., Kelly, M. W., Lessard, K. N., Khalil, A. and Henry, C. A. (2010). Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis. Dev Biol 344(2): 809-826.
  2. Goody, M. F., Kelly, M. W., Reynolds, C. J., Khalil, A., Crawford, B. D. and Henry, C. A. (2012). NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy. PLoS Biol 10(10): e1001409.

简介

荧光共轭的鬼笔环肽是允许F-肌动蛋白可视化的染色剂。 在免疫组织化学中,一抗和荧光共轭二抗可用于显现亚细胞定位和目标蛋白的相对量。 这是一个协议为Phalloidin和抗体染色的斑马鱼胚胎5天和更年轻。

关键字:肌肉, 交接处, 抗体

材料和试剂

  1. Alexa Fluor 488或546 Phalloidin(Life Technologies)
  2. 所需的一级抗体(参见表1,常用于Henry实验室的抗体信息)
  3. Alexa Fluor 488,546或633次级抗体(例如山羊抗小鼠或山羊抗兔二抗,Life Technologies)。
  4. 真空润滑脂(例如,Dow Corning高真空润滑脂)
  5. 10x PBS(请参阅配方)
  6. PBS 0.1%Tween-20 (参见配方)
  7. PBS 2%Tween-20 ®(见配方)
  8. 8%PFA(见配方)
  9. 4%PFA(参见配方)
  10. 块(参见配方)
  11. 80:20甘油:PBS溶液(见配方)

设备

  1. 两个精细镊子(例如 。 Dumont#5镊子)
  2. 两个解冻工具(例如,昆虫针超级粘在玻璃毛细管的末端,图1)
  3. 长凳摇椅
  4. 1.5-2ml微量离心管
  5. 玻璃巴斯德移液器
  6. 移液泵
  7. 微量移液器
  8. 微量吸头
  9. 显微镜载玻片
  10. 显微镜载玻片
  11. 解剖显微镜
  12. 用于图像采集的显微镜(例如运行AxioVision软件的Zeiss Axio Imager)


    图1. Deyolking工具。Deyolking工具可用于手术 从固定的斑马鱼胚胎中删除卵黄囊。 液压工具 包括昆虫针(Fine Science Tools,目录号:26002-20) 超级胶合到玻璃毛细管的末端(Sutter Instruments, 目录号:BF100-50-10)。 然后包裹玻璃毛细管   在实验室磁带。

程序


A.固定和鬼笔环肽染色:

  1. Dechorionate胚胎用两对精细的镊子。在解剖显微镜下,用握在你的手中的一双镊子夹住胚胎的绒毛。用另一只手中的镊子,捏住附近的原始捏和的绒毛,轻轻撕开绒毛分离你的手。用镊子重复挤压和撕裂的绒毛,直到胚胎去除细胞
  2. 使用移液管泵和玻璃移液管,转移dechorionated胚胎到微量离心管。在一个管中最多放置10个胚胎。相应地标记管。
    注意:移液器泵和玻璃移液管吸头可用于本方案中的所有溶液添加和去除,不包括添加一级和二级抗体。用玻璃移液管吸头上下移动胚胎,每次更换溶液可改善洗涤效果,避免胚胎粘在一起。。
  3. 从微量离心管中尽可能多地除去液体。
  4. 在使用PFA时戴上手套。向每个微量离心管中加入约0.5ml的4%PFA
  5. 将含有固定剂的微量离心管在室温下置于台式摇床上(轻轻摇动)4小时或4℃过夜。使管垂直于摇摆运动定向,使得胚胎在管中从一侧到另一侧摇摆,而不是从顶部到底部摇摆。
  6. 取出固定剂,并将其丢弃在适当的废物容器中。
  7. 冲洗胚胎3次,每次5分钟在〜0.5ml PBS 0.1%Tween-20中。在本协议中的每次冲洗中,轻轻地将胚胎重悬在溶液中以改善冲洗并防止它们粘在一起。
  8. 除去最后的PBS 0.1%Tween-20冲洗并向每个管中加入〜0.5ml PBS 2%TritonX-100以透化胚胎用于鬼笔环肽染色。
  9. 在其侧面放置含有PBS 2%Trition的管,并在室温下轻轻摇动1.5小时
  10. 取出PBS 2%TritonX-100 ,并使用P20微量吸管添加19μl的PBS 2%TritonX-100 到每个管。使用P20微量移液管,每管加入1μlAlexa Fluor 488或546鬼笔环肽(在使用鬼笔环肽时戴手套)。
  11. 放置管的侧面,轻轻摇动(如果可能)在4°C过夜。从这一刻起,尽可能将管保持在黑暗中(例如 )。


B.用单克隆/多克隆抗体染色的抗体:

  1. 从管中除去鬼笔环肽,并向每个管中加入约0.5ml的块
  2. 在室温下,在黑暗中至少1小时,将含有块的岩石管放在其侧面。
  3. 使用微量移液管添加块和一抗溶液到每个管,以获得适当的一抗稀释为斑马鱼胚胎染色优化。 初级抗体在块中的1:100稀释是开始的好地方。 初级抗体稀释度可以为1:10至1:5,000。 在加入一抗之前,一些抗体可能需要另外的透化步骤(例如在-20℃下在甲醇或丙酮中孵育,蛋白酶处理)。 一些一抗可能不与鬼笔环肽染色相容。
  4. 将胚胎在初级抗体中在4℃下在黑暗中用管子在其侧面孵育过夜,如果可能,轻轻摇动。
  5. 取出一抗稀释液,加入〜0.5 ml块
  6. 在黑暗中将胚胎在其侧面进行块胚胎,并在室温下轻轻摇动〜8小时。如果需要减少非特异性背景染色,可以加入另外的漂洗液
  7. 除去块,并使用微量移液器添加199微升抗体块和1微升适当的荧光共轭二抗每个管。注意在确定使用适当的二级抗体时用于鬼笔环肽染色的荧光团的波长。确保使用的二抗对应于一抗是单克隆抗体还是多克隆抗体
  8. 孵育胚胎在这种二次抗体稀释过夜在4℃在黑暗中用管在他们的边,轻轻摇摆,如果可能的话。
  9. 去除二次抗体稀释,并在黑暗中将胚胎储存在〜0.5ml PBS 0.1%Tween-20中。如果需要减少非特异性背景染色,可以加入在PBS 0.1%Tween-20中的额外漂洗。


C.损伤,安装和成像:

  1. 手动脱脂胚胎在1x PBS与两个解冻工具。使用解冻工具来定向胚胎侧面。通过将一只手握住的解冻工具的尖端插入卵黄囊和胚胎体之间的位置来保持胚胎。另一方面,使用解冻工具来"剪切"卵黄囊远离胚胎的身体,同时保持另一只手静止。一旦大部分卵黄已经被去除,使用根据需要清理和删除任何额外的卵黄囊残余。吮吸和丢弃卵黄囊的片断。重复脱脂过程,直到所有胚胎脱脂。
  2. 转移胚胎到80:20甘油:PBS。如果胚胎在转移至80:20甘油:PBS时脱水,则可能必须实施递增浓度的甘油的缓慢洗涤。例如,在转移至80:20甘油:PBS之前,可能需要在30:70甘油:PBS和60:40甘油:PBS各自中进行1小时长的洗涤。使用一个脱脂的胚胎作为测试样品,并将其从1×PBS移至80:20甘油:PBS。如果胚胎的形状改变,继续上面列出的其余胚胎的孵化
  3. 点玻璃显微镜幻灯片与4个小点的真空油脂,使点将支撑正方形盖玻片的四个角。
  4. 转移一个胚胎和两滴80:20甘油:PBS在显微镜载玻片上的4个点的润滑脂的中间。
  5. 用解冻工具定位胚胎,使其侧面安装并平整。
  6. 将盖玻片放在样品上,轻轻地向下压在盖玻片的角落,直到它接触胚胎,但不压扁它。
  7. 重复步骤C3-6,直到所有胚胎安装。保持你准备好的幻灯片在幻灯片在黑暗中4℃,直到他们成像。图像你染色的斑马鱼胚胎在〜2周内,越早越好。代表图像如图2所示。


    图2.鬼笔环肽染色的代表性结果。(A)解冻的,侧面安装的斑马鱼胚胎,前左,背顶的明场图像。 (B)显示鬼笔环肽546染色的相同胚胎的荧光显微照片。 (C)用运行AxioVision软件的Zeiss Axio Imager上的20x物镜拍摄的相同胚胎的图像。鬼笔环肽546染色使得骨骼肌纤维的肌动蛋白细胞骨架可视化。关于抗体染色的代表性图像,请参考(Goody等人,2010)。

    表1. Henry实验室常用抗体的抗体染色信息。

    名称 公司
    产品#
    稀释
    单/多克隆
    合作 鬼笔环肽®
    额外


    渗透性®

    β-半胱氨酸聚糖
    Novocastra
    NCLbDG
    1:50
    单克隆

    没有
    F59
    发展研究 杂交瘤银行(DSHB)

    1:10
    单克隆

    没有
    纤连蛋白
    Sigma
    F3648
    1:50
    多克隆

    没有
    层粘连蛋白-111 Thermo Scientific
    RB-082-A0
    1:50
    多克隆

    没有
    桩蛋白
    BD转导 实验室
    610051
    1:50
    单克隆

    没有
    FAK pY397或pY861
    Invitrogen

    1:50
    多克隆

    没有
    营养不良蛋白
    Sigma
    D8043
    1:50
    单克隆

    没有
    MF20
    DSHB

    1:10
    单克隆
    不太好
    没有
    F310
    DSHB

    1:10
    单克隆
    不太好
    没有
    Vinculin
    Sigma
    V4505
    1:10
    单克隆
    未知
    PBS 2%TritonX-100在室温下培养2.5小时
    4D9 /雕刻
    DSHB

    1:2
    单克隆

    10分钟 在-20℃
    β-联蛋白
    Abcam
    Ab6302
    1:500
    单克隆
    未知
    需要特殊 固定剂(4%PFA,4%蔗糖,3mM CaCl 2,1×PBS)
    GFP
    分子探针
    A21311
    1:200
    多克隆
    未知
    2小时固定在4%PFA中 在室温下

食谱

  1. 磷酸盐缓冲盐水(PBS)
    1. 10x PBS
      加入1升瓶
      74克NaCl
      19.4g Na 2 HPO 4 7Hl 2 O·
      4.37g NaH 2 PO 4·H 2 O 2·h / 〜800ml dH 2 O
      搅拌至溶解。 用dH 2 O使体积达到1L。 高压灭菌。 稀释10x PBS至2x PBS或1x PBS与dH2O。 在室温下储存。
    2. PBS 0.1%Tween-20 ®
      向1 L 1x PBS中加入
      1 ml Tween-20 ®
      混合溶液。 在室温下储存。
    3. PBS 2%TritonX-100 ®
      向1 L 1x PBS中加入
      20ml TritonX-100 ®
      混合溶液。 在室温下储存。
  2. 多聚甲醛(PFA)
    1. 8%PFA
      加入50ml锥形管
      4 g PFA
      30ml dH 2 O 2/20滴1N NaOH,
      轻轻加热并搅拌直至溶解。 用dH 2 O使体积达到50ml。 通过#1 Whatman纸过滤溶液。 加入20滴1N HCl并混合。 储存于4°C。 在1周内使用。
    2. 4%PFA
      加入50ml锥形管
      25ml 8%PFA
      25ml 2×PBS
      混合,然后储存在4°C。 在1周内使用。
  3. 阻止
    加入到50ml锥形管中 2.5g牛血清白蛋白(BSA)
    40 ml 1x PBS
    轻轻加热并搅拌直至BSA溶解。 然后添加
    0.5ml DMSO 0.5ml TritonX-100 ®
    0.1克皂苷
    用1x PBS加至50 ml。 储存于4°C。 在1周内使用。
  4. 80:20甘油:PBS
    加入到50ml锥形管中 40毫升甘油 10ml 1x PBS
    混合溶液。 在室温下贮存

致谢

此协议改编自以前的出版物:Goody等人(2010)和Goody等人(2012)。 该协议的开发由NIH授予RO1HD052934-01A1至CAH支持。 MFG感谢缅因大学生物医学科学与工程研究生院的资助。

参考文献

  1. Goody,M.F.,Kelly,M.W.,Lessard,K.N.,Khalil,A.and Henry,C.A。(2010)。 Nrk2b介导的NAD + 产生调节细胞粘附,是肌肉所需的 形态发生在体内:Nrk2b和NAD +在肌肉形态发生中。 344(2):809-826。
  2. Goody,M.F.,Kelly,M.W.,Reynolds,C.J.,Khalil,A.,Crawford,B.D.and Henry,C.A。(2012)。 NAD + 生物合成改善了肌肉营养不良的斑马鱼模型。 PLoS Biol 10(10):e1001409。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Goody, M. F. and Henry, C. A. (2013). Phalloidin Staining and Immunohistochemistry of Zebrafish Embryos. Bio-protocol 3(11): e786. DOI: 10.21769/BioProtoc.786.
  2. Goody, M. F., Kelly, M. W., Reynolds, C. J., Khalil, A., Crawford, B. D. and Henry, C. A. (2012). NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy. PLoS Biol 10(10): e1001409.
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garima tripathi
kims
how to use Alexa Fluor 488,?
what is dilution and how to dilute for ready to use?
11/18/2014 9:01:38 PM Reply
Michelle F. Goody
Graduate School of Biomedical Sciences and Engineering, University of Maine, USA

We buy phalloidin from Life Technologies and dissolve it in 1.5 mL MeOH. We then aliquot and freeze the phalloidin stock at -20 C. Before incubating the zebrafish embryos in phalloidin, it is diluted 1 part phalloidin to 19 parts PBS with 2% Triton-X 100.

11/21/2014 12:23:45 PM


garima tripathi
kims

PBS with 2 % trinton X100 menas? can you elaborate

11/21/2014 2:34:52 PM


Michelle F. Goody
Graduate School of Biomedical Sciences and Engineering, University of Maine, USA

There is a recipe for PBS with 2% TritonX 100 in the protocol above.

11/21/2014 6:11:41 PM