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Collecting and Fixing Nuclear GFP/RFP in L1 Larva for Imaging
用来拍照的转化GFP/RFP的幼期蠕虫的收集和固定方法   

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Abstract

In this protocol, L1 stage larvae are collected that carry nuclear-localized GFP/wCherry reporters. These can be fixed so that the GFP/wCherry maintains nuclear localization and stain nuclei by DAPI. This protocol therefore achieves the collection and fixation of nuclei in worm L1 larvae.

Materials and Reagents

  1. Acetone
  2. Formaldehyde
  3. DAPI
  4. Poly-lysine
  5. Glycerol
  6. Cytoseal 280 (Richard-Allan Scientific, catalog number: 8311-4 )
  7. Used Qiaquick Spin Column (QIAGEN)
  8. 11.58 μm glassbeads (Whitehouse Scientific, catalog number: MS0012 )
  9. KCl
  10. NaCl
  11. Na2EGTA
  12. Triton X-100
  13. EDTA
  14. PIPES
  15. 2x modified MRWB (see Recipes)
  16. DAPI staining solution (see Recipes)
  17. M9 buffer (see Recipes)
  18. Tris triton buffer (TTB) (see Recipes)

Equipment

  1. Standard bench top microcentrifuge
  2. 16-slide glass staining jar (Thermo Fisher Scientific, catalog number: 08-810 )
  3. Spatula
  4. Microscope
  5. Glass container
  6. Glass coverslip
  7. Glass slide
  8. 18 x 18 mm glass cover slip
  9. 25 x 75 mm glass slide

Procedure

  1. Preparing larva
    1. Begin with a plate that contains many eggs (100+)
    2. Use the spatula to remove any chunks which many retain worms. Be sure to flame between uses so no worms are transferred between plates.
    3. Using a spatula, carefully displace and remove the agar from the plastic plate. Place the agar in a 16-slide glass-staining jar keeping the surface with worms facing upwards.
    4. Rinse the agar in the glass container three times with deionized water, taking care that water does not directly hit the agar.
    5. Using the spatula, place the agar back into the plastic container. Remember which side faces upward!
    6. Check under the microscope to make sure no worms are left on the plate. If worms are left, repeat rinses until no worms are left. There should be plenty of eggs (100+).
    7. Leave the plate at room temperature (RT) (25 °C) for 2 h.
    8. Check that L1 larva has emerged.

  2. Freezing and Fixing the Worms
    1. Wash the plate with 500 μl M9 buffer and transfer to 1.5 ml centrifuge tube. Repeat twice.
    2. Spin 3,000 rpm, 2 min. Remove supernatant taking care not to disturb worms at the bottom. Resuspend with 1 ml M9.
    3. Spin 3,000 rpm, 2 min. Remove supernatant taking care not to disturb worms at the bottom. Resuspend with 500 μl M9.
    4. Transfer to a used Qiaquick Spin Column. Spin 3,000 rpm, 2 min with lid open.
    5. Close Qiaquick column cap and place column and collection tube separated into a bucket. Add liquid nitrogen. The next few steps should be performed as quickly as possible after liquid nitrogen is added.
    6. Add 200 μl acetone (-20 °C) to the column and immediately spin 2,000 rpm, 30 sec.
    7. Add 200 μl acetone (-20 °C) to the column and place in -20 °C freezer for 1 min. Then spin 2,000 rpm 30 sec.
    8. Add 200 μl fresh MWRB/formaldehyde solution (50% 2x modified MRWB, 5% formaldehyde = 100 μl 2x modified MRWB, 100 μl 10% formaldehyde) and let sit at RT for 1 h. Then spin at 2,000 rpm, 30 sec.
    9. Add 200 μl TTB and spin 2,000 rpm, 30 sec. Repeat to remove all formaldehyde.
    10. Add 200 μl DAPI staining solution. Let sit at RT for 1 h. Spin 2,000 rpm, 30 sec.
    11. Add 200 μl TTB and pipette up and down to resuspend the worms in the solution. Transfer to 1.5 ml centrifuge tube to collect worms.

  3. Preparing the slides
    1. Add 75 μl of .5% poly-lysine (in H2O) to a 18 x 18 mm glass cover slip. Cover using plastic dish lid and let sit at RT for at least 30 min.
    2. Recollect excess poly-lysine.
    3. Wash cover slip using distilled H2O and air dry.
    4. Add a drop of well suspended 11.58 μm glass beads (in acetone) onto the treated surface of a 25 x 75 mm glass slide. Air dry.

  4. Mounting the Worms
    1. Add 20 μl of worms in TTB to the poly-lysine treated side of the coverslip. Leave at RT for 30 min to allow worms to stick to the coverslip.
    2. Remove as much TTB as possible but observe this removal step under the microscope to make sure most worms are stuck to the poly-lysine.
    3. Add 75 μl 50% glycerol to the coverslip. Carefully remove glycerol from the sides. Add 50 μl 50% glycerol to the middle of the coverslip. This helps to disperse an excess TTB, making the drop closer to 50% glycerol. Remove excess liquid from the sides.
    4. Place slide over the cover slip and seal with Cytoseal 280.
    5. Place in a slide holder and refrigerate until use.

Recipes

  1. 2x modified MRWB
    160 mM KCl
    40 mM NaCl
    20 mM Na2EGTA
    10 mM Spermidine HCl
    30 mM PIPES (pH 7.4)
  2. TTB (Tris triton buffer)
    100 mM Tris-HCl (pH 7.4)
    1% Triton X-100
    1 mM EDTA
  3. M9 buffer
    Refer to: common worm media and buffers
  4. DAPI staining solution
    100 μl M9
    100 μl deionized H2O
    0.1 μl 1 mg/ml DAPI

Acknowledgments

This protocol has been adapted from Rigaut et al. (1999) and Puig et al. (2001).

References

  1. Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M. and Seraphin, B. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24(3): 218-229.
  2. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M. and Seraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17(10): 1030-1032.

简介

在这个协议,收集L1阶段幼虫携带核定位GFP/wCherry记者。 这些可以是固定的,使GFP/wCherry保持核定位和染色核由DAPI。 因此该协议实现了在L1幼虫中核的收集和固定。

材料和试剂

  1. 丙酮
  2. 甲醛
  3. DAPI
  4. 聚赖氨酸
  5. 甘油
  6. Cytoseal 280(Richard-Allan Scientific,目录号:8311-4)
  7. 二手Qiaquick旋转柱(QIAGEN)
  8. 11.58μm玻璃珠(Whitehouse Scientific,目录号:MS0012)
  9. KCl
  10. NaCl
  11. Na 2 EGTA
  12. Triton X-100
  13. EDTA
  14. PIPES
  15. 2x修改的MRWB(参见配方)
  16. DAPI染色溶液(见配方)
  17. M9缓冲区(请参阅配方)
  18. Tris triton缓冲液(TTB)(参见配方)

设备

  1. 标准台式微量离心机
  2. 16-载玻片染色罐(Thermo Fisher Scientific,目录号:08-810)
  3. 小铲
  4. 显微镜
  5. 玻璃容器
  6. 玻璃盖玻片
  7. 玻璃片
  8. 18 x 18毫米玻璃盖玻片
  9. 25 x 75毫米玻璃滑轨

程序

  1. 准备幼虫
    1. 从包含许多鸡蛋(100+)的板子开始
    2. 使用刮铲去除任何大块留下蠕虫的块。 确保在使用之间火焰,所以没有蠕虫在板之间传输
    3. 使用刮铲,小心地从塑料板中取代和去除琼脂。 将琼脂放在16片玻璃染色罐中,保持表面有虫向上。
    4. 用去离子水冲洗玻璃容器中的琼脂三次,注意水不会直接碰到琼脂。
    5. 使用刮铲,将琼脂放回塑料容器中。 记住哪一面朝上!
    6. 在显微镜下检查,以确保没有蠕虫留在板上。 如果留下了蠕虫,请重复冲洗,直到没有蠕虫留下。 应该有大量的鸡蛋(100+)。
    7. 将板在室温(RT)(25℃)下放置2小时
    8. 检查L1幼虫是否出现。

  2. 冷冻和修复蠕虫
    1. 用500μlM9缓冲液洗板,转移到1.5 ml离心管中。 重复两次。
    2. 旋转3,000rpm,2分钟。 除去上清液,小心不要扰动底部的蠕虫。 用1ml M9重悬
    3. 旋转3,000rpm,2分钟。 除去上清液,小心不要扰动底部的蠕虫。 用500μlM9重悬
    4. 转移到使用的Qiaquick旋转柱。 旋转3,000rpm,盖子打开2分钟。
    5. 关闭Qiaquick柱盖,将柱子和收集管分成桶。 加入液氮。 在加入液氮后,应尽快进行下几个步骤
    6. 加200μl丙酮(-20°C)到立柱,立即旋转2000 rpm,30秒。
    7. 加入200μl丙酮(-20°C)到该柱,并置于-20°C冰箱1分钟。 然后旋转2000 rpm 30秒。
    8. 加入200μl新鲜的MWRB /甲醛溶液(50%2x改性MRWB,5%甲醛=100μl2×改性MRWB,100μl10%甲醛),并在室温下放置1小时。 然后以2,000rpm,30秒转速。
    9. 加入200微升TTB和旋转2000 rpm,30秒。 重复以去除所有甲醛。
    10. 加入200μlDAPI染色溶液。 让我们在室温下放置1小时。 旋转2000 rpm,30秒。
    11. 添加200微升TTB和吸管向上和向下重新悬浮在溶液中的蠕虫。 转移到1.5ml离心管收集蠕虫

  3. 准备幻灯片
    1. 将75μl的0.5%聚赖氨酸(在H 2 O中)加入到18×18mm玻璃盖玻片中。 盖上塑料盘盖,并放置在室温至少30分钟。
    2. 回收多余的聚赖氨酸。
    3. 使用蒸馏水H 2 O洗涤盖玻片并风干
    4. 将一滴充分悬浮的11.58μm玻璃珠(在丙酮中)加到25×75mm载玻片的处理表面上。 空气干燥。

  4. 安装蠕虫
    1. 添加20微升的蠕虫在TTB到盖玻片的聚赖氨酸处理的一面。 在室温下放置30分钟,使蠕虫粘在盖玻片上。
    2. 删除尽可能多的TTB,但在显微镜下观察这个删除步骤,以确保大多数蠕虫被粘在聚赖氨酸。
    3. 加盖75微升50%甘油到盖玻片。 小心从侧面去除甘油。 加入50微升50%甘油到盖玻片的中间。 这有助于分散过量的TTB,使得液滴更接近50%甘油。 从侧面清除多余的液体。
    4. 将盖玻片放在盖玻片上,并用Cytoseal 280密封。
    5. 放置在玻片保持器中并冷藏,直到使用。

食谱

  1. 2x修改的MRWB
    160 mM KCl
    40 mM NaCl
    20mM Na 2 EGTA
    10mM盐酸精胺
    30mM PIPES(pH7.4)
  2. TTB(Tris triton buffer)
    100mM Tris-HCl(pH7.4) 1%Triton X-100 1mM EDTA
  3. M9缓冲区
    请参阅:常见蠕虫媒介和缓冲区
  4. DAPI染色溶液
    100μlM9
    100μl去离子H 2 O 2 / 0.1μl1mg/ml DAPI

致谢

该协议已经改编自Rigaut等人(1999)和Puig等人(2001)。

参考文献

  1. Puig,O.,Caspary,F.,Rigaut,G.,Rutz,B.,Bouveret,E.,Bragado-Nilsson,E.,Wilm,M.and Seraphin,B。 串联亲和纯化(TAP)方法:蛋白复合物纯化的一般程序。 方法 24(3):218-229
  2. Rigaut,G.,Shevchenko,A.,Rutz,B.,Wilm,M.,Mann,M。和Seraphin,B。(1999)。 蛋白质复合物表征和蛋白质组探索的通用蛋白质纯化方法 Biotechnol 17(10):1030-1032
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, X. (2012). Collecting and Fixing Nuclear GFP/RFP in L1 Larva for Imaging. Bio-protocol 2(5): e94. DOI: 10.21769/BioProtoc.94.
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