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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.

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[Bio101] Oil Red O Staining of Fixed Worm

Cell Biology > Cell staining > Lipid

[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, C elegans, Fat stores

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.


How to cite this protocol: He, F. (2012). Oil Red O Staining of Fixed Worm. Bio-protocol Bio101: e230. DOI: 10.21769/BioProtoc.230; Full Text



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12/7/2015 6:42:47 AM  

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

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