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[Bio101] Oil Red O Staining of Fixed Worm
[Bio101] 固定蠕虫的油红染色   

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Abstract

Oil red O staining is used to assess major fat stores in C. elegans. This protocol is adapted from the Ashrafi Lab at the University of California-San Francisco.

Keywords: Lipid staining(脂质染色), C elegans(C线虫), Fat stores(脂肪储存)

Materials and Reagents

  1. 20% paraformaldehyde
  2. Na2EGTA
  3. Spermidine 3 HCl (1 g) (Sigma-Aldrich, catalog number: 85578 )
  4. Spermine (5 g) (Sigma-Aldrich, catalog number: 85590 )
  5. Oil Red O (Sigma-Aldrich, catalog number: O0625 )
  6. NaPIPES (pH 7.4)
  7. Beta-ME
  8. Isopropanol
  9. DTT
  10. Tris Base
  11. HCl
  12. PBS
  13. Agarose
  14. NaCl
  15. KCl
  16. Na2HPO4•7H2O
  17. KH2PO4
  18. NaOH
  19. 10x PBS buffer (see Recipes)
  20. 1 M Tris-Cl (pH 7.4) (see Recipes)
  21. 2x MRWB (see Recipes)
  22. Reduction buffer (see Recipes)

Equipment

  1. Eppendorf Thermomixer Shaker (Eppendorf, catalog number: EF4283A )
  2. Dissecting stereo microscope (LEICA MZ12 )
  3. Compound microscope (Nikon ECLIPSE, model: E600 )
  4. Tabletop centrifuge
  5. 15 ml conical tube
  6. 1.5-ml Eppendorf tube
  7. 0.2 µM filter

Procedure

  1. Collect worms at desired stage with 1x PBS and transfer to 15 ml conical tube. Allow the worms to settle by gravity if using adults or spin down larval stage worms at 1,000 x g for 30 sec.
    Note: Make sure to collect enough worms (~200-500) some will be lost during the procedure.
  2. Aspirate supernatant and wash again with 10 ml 1x PBS. Allow to settle by gravity or spin as described at step 1.
  3. After second wash, discard most of the supernatant except 400 µl and transfer it to 1.5-ml Eppendorf tube.
  4. For fixation, add 500 µl 2x MRWB (freshly made) and 100 µl 20% paraformaldehyde to the tube containing 400 µl sample in 1x PBS.
  5. Mix the solution by gently inverting the tube and fix 30 min at RT with gently shaking (1,000 rpm) on an Eppendorf Thermomixer Shaker (invert the tube every 5-8 min without shaking).
  6. Wash twice with 1 ml Tris-Cl buffer (100 mM, pH 7.4). Between wash, spin down worms using a tabletop centrifuge at 1,500 x g for 30 sec.
  7. After 2nd wash, discard supernatant except of 100 µl and add 900 μl of reduction buffer. Mix by gently inverting the tube and reduce 30 min at RT with gently shaking.
  8. Pellet worms by centrifuging at 1,500 x g for 30 sec.
  9. Wash with 500 µl 1x PBS; spin down at 1,500 x g for 30 sec.
  10. Aspirate to 300 µl, add 700 µl isopropanol. Mix by gently inverting the tube and leave at RT 15 min with gentle shaking.
  11. Spin at 1,500 x g for 30 sec to collect.
  12. Suck off all isopropanol and add Oil Red O dye solution.
  13. Preparation of Oil Red O solution, 0.5 g Oil Red O in 100 ml anhydrous isopropanol,
    Equilibrate it for two days by stirring at RT.
  14. About 15 min before use, mix 4 vol ddH2O with 6 vol dye solution and leave at RT for 15 min. It appears cloudy.
  15. Filter the solultion with 0.2 µM pore size filter unit. Effluent should appear clear.
  16. Add 1 ml of the 60% filtered dye to the worms. Leave on Thermomixer Shaker (700 rpm shaking or a rotator) overnight at RT.
  17. Spin at 1,200 x g for 30 sec to pellet worms.
  18. Such off as much dye as possible and wash once with 1x PBS.
  19. Mount directly on a glass slide with an agarose pad (dissolve 2% agarose in 1x PBS).
    Note: Keep slides in a humidity container to prevent samples for drying out.

Recipes

  1. 500 ml 10x PBS buffer
    40 g NaCl
    1 g KCl
    5.75 g Na2HPO4•7H2O
    1 g KH2PO4
    Dissolve in 400 ml ddH2O and adjust to pH 7.4 with HCl or NaOH. Bring volume to 500 ml with ddH2O.
  2. 100 ml 1 M Tris-Cl (pH 7.4)
    Dissolve 12.114 g Tris Base in 80 ml ddH2O and adjust to pH 7.4 with concentrated HCl. Bring volume to 100 ml with ddH2O.
  3. 2x MRWB
    160 mM KCl
    40 mM NaCl
    14 mM Na2EGTA
    1 mM Spermidine HCl
    0.4 mM Spermine
    30 mM NaPIPES (pH 7.4)
    0.2% beta-ME
  4. Reduction buffer
    100 mM Tris-Cl (pH 7.4)
    10 mM DTT (1.54 mg in 1 ml)

Acknowledgments

This protocol is adapted from the Ashrafi Lab at the University of California San Francisco.

References

  1. O'Rourke, E. J., Soukas, A. A., Carr, C. E. and Ruvkun, G. (2009). C. elegans major fats are stored in vesicles distinct from lysosome-related organelles. Cell Metab 10(5): 430-435.

简介

油红O染色用于评价主要脂肪储存在C中。 elegans 。 该协议改编自加州大学旧金山分校的Ashrafi实验室。

关键字:脂质染色, C线虫, 脂肪储存

材料和试剂

  1. 20%多聚甲醛
  2. Na 2 EGTA
  3. 亚精胺3 HCl(1g)(Sigma-Aldrich,目录号:85578)
  4. 精胺(5g)(Sigma-Aldrich,目录号:85590)
  5. 油红O(Sigma-Aldrich,目录号:O0625)
  6. NaPIPES(pH 7.4)
  7. Beta-ME
  8. 异丙醇
  9. DTT
  10. Tris碱
  11. HCl
  12. PBS
  13. 琼脂糖
  14. NaCl
  15. KCl
  16. Na 2 HPO 4 4 7H 2 O O 2 /
  17. KH 2 PO 4
  18. NaOH
  19. 10x PBS缓冲液(见配方)
  20. 1 M Tris-Cl(pH 7.4)(参见配方)
  21. 2x MRWB(请参阅配方)
  22. 还原缓冲区(请参阅配方)

设备

  1. Eppendorf Thermomixer Shaker(Eppendorf,目录号:EF4283A)
  2. 解剖立体显微镜(LEICA MZ12)
  3. 复合显微镜(Nikon ECLIPSE,型号:E600)
  4. 台式离心机
  5. 15 ml锥形管
  6. 1.5 ml Eppendorf管
  7. 0.2μM滤波器

程序

  1. 收集蠕虫在所需的阶段用1x PBS和转移到15毫升锥形管。 如果使用成人或者在1000英尺/秒下旋转幼虫阶段蠕虫30秒,则允许蠕虫通过重力沉降。
    注意:请务必收集足够的蠕虫(〜200-500),否则会在手术过程中丢失。
  2. 吸出上清液,并再次用10ml 1x PBS洗涤。 允许通过重力或旋转沉降,如步骤1所述。
  3. 第二次洗涤后,弃去大部分上清液,除了400μl,并将其转移到1.5ml Eppendorf管
  4. 对于固定,添加500微升2×MRWB(新鲜)和100微升20%多聚甲醛到含有400微升样品在1×PBS的管。
  5. 混合溶液,轻轻倒转试管,并在Eppendorf Thermomixer振荡器上轻轻摇动(1000 rpm),在室温下固定30分钟(每5-8分钟振荡试管,不摇动)。
  6. 用1ml Tris-Cl缓冲液(100mM,pH 7.4)洗涤两次。 在洗涤之间,使用台式离心机以1,500×g离心蠕虫30秒。
  7. 2次洗涤后,弃去100μl上清液,加入900μl还原缓冲液。 通过轻轻颠倒试管混合,并在室温下轻轻摇动30分钟
  8. 通过在1,500×g离心30秒来离心颗粒蠕虫。
  9. 用500μl1×PBS洗涤; 向下旋转1500英尺x g 30秒。
  10. 吸出到300μl,加入700μl异丙醇。 通过轻轻颠倒试管混合,并在室温下温和振荡15分钟
  11. 旋转1500英尺x g 30秒即可收集
  12. 吸掉所有异丙醇并加入油红O染料溶液
  13. 油红O溶液,0.5g油红O在100ml无水异丙醇中的制备 通过在室温下搅拌将其平衡两天
  14. 使用前约15分钟,用6体积的染料溶液混合4体积ddH 2 O,并在室温下放置15分钟。 它看起来多云。
  15. 用0.2μM孔径过滤器过滤该溶质。 流出物应清晰。
  16. 加入1毫升60%过滤染料的蠕虫。 在室温下放置在Thermomixer摇床(700rpm摇动或旋转器)上过夜
  17. 旋转1,200英尺x g 30秒,以沉淀蠕虫。
  18. 尽可能多的染料,并用1x PBS洗一次。
  19. 用琼脂糖垫直接装在载玻片上(溶解1%PBS中的2%琼脂糖) 注意:将载玻片放在湿度容器中,以防止样品变干。

食谱

  1. 500ml 10x PBS缓冲液
    40克NaCl
    1克KCl
    5.75g Na 2 HPO 4 7H 2 O 2 1 g KH sub 2 PO 4 sub
    溶解在400ml ddH 2 O中,并用HCl或NaOH调节至pH 7.4。 用ddH 2 O将体积调至500ml。
  2. 100 ml 1M Tris-Cl(pH7.4) 将12.114g Tris碱溶解在80ml ddH 2 O中,并用浓HCl调节至pH 7.4。 用ddH 2 O将体积加至100ml。
  3. 2x MRWB
    160 mM KCl
    40 mM NaCl
    14mM Na 2 EGTA
    1mM盐酸精胺
    0.4mM精胺
    30mM NaPIPES(pH7.4) 0.2%β-ME
  4. 减少缓冲区
    100mM Tris-Cl(pH7.4) 10mM DTT(1.54mg,在1ml中)

致谢

该协议改编自加州大学旧金山分校的Ashrafi实验室。

参考文献

  1. O'Rourke,E.J.,Soukas,A.A.,Carr,C.E.and Ruvkun,G。(2009)。 C。 elegans 主要脂肪存储在与溶酶体相关的细胞器不同的囊泡中。细胞Metab 10(5):430-435。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, F. (2012). Oil Red O Staining of Fixed Worm. Bio-protocol Bio101: e230. DOI: 10.21769/BioProtoc.230;
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David Buj
University of Huddersfield
Hello,

Could you explain the purpose of the MRWB buffer? Some papers use it or PBS indistinguishly. Why use spermine and spermidine?

Why is the reduction buffer necessary? It is not used by O'Rourke (O'Rourke et al., 2009)

Thank you.

Regards

David Buj
PhD student
University of Huddersfield



O'Rourke, E. J., Soukas, A. A., Carr, C. E. and Ruvkun, G. (2009). C. elegans major fats are stored in vesicles distinct from lysosome-related organelles. Cell Metab 10(5): 430-435
12/7/2015 6:42:47 AM Reply