Staining of cells

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Staining of Lipid Droplets with Oil Red O.
Preparation of Oil Red O
1. Prepare a 1% solution of oil Red O in propan-2-ol (typically 0.6g of oil Red O in 6ml propan-2-ol is sufficient). Heat to 37oC to dissolve (which takes about 20-30min).
2. Before cells are ready for staining, add 4ml H2O to 6ml of oil Red O solution (i.e. makes the solution 0.6% oil red O in 60% propan-2-ol).
3. To remove the precipitate, filter through a 0.45um filter with a syringe (do not use excess pressure!). The filtrate is the final staining solution.

Staining Cells with Oil Red O
The method below assumes that cells have been seeded on 13mm coverslips and have been cultivated in 24-well nunc plates.
1. Rinse coverslips with 1ml PBS and aspirate off the PBS.
2. Add 0.5 ml 60% propan-2-ol briefly and aspirate (to equilibrate the coverslips with solvent).
3. Add 0.5ml oil Red O staining solution and leave at room temperature for 1.30-1.45min then aspirate.
4. Add 0.5 ml 60% propan-2-ol briefly and aspirate (to remove excess stain).
5. Rinse coverslips 3 times with 1ml PBS and aspirate between each rinse.
6. Mount coverslips (do not use Citifluor if possible or any mounting fluid that contains alcohols or glycerol since it can lead to fusion of droplets).

see discussion in "Staining of lipid droplets with oil Red O"

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