Dear Leah, I have checked the Histonet archives and  a 'histonetter' Gayle Callis mentioned that in her opinion the best method for Oil red O staining on  4%PFA fixed frozen sections was one described by Charukian in Journal of Histotechnology. It might be worth trying to find this paper. Good luck with your project.

We have has good results with the Lillie& Ashburn method of 1942:

Tissue preparation:

Use a dewar flask which has liquid nitrogen in it. Then lower a small metal beaker hooked on a stand and which contains isopentane in to the liquid nitrogen. You can place your muscle on a piece of cork before freezing with OCT to stabilize. When the liquid nitrogen is cold, lower your muscle in to it.  If  you do not freeze your muscle right you will get ice crystal artifact.  You can then section your material in the cryostat.(see the book: Muscle Biopsy by Victor Dubowitz)

Stain

Stock solution: Saturated oil red o solution made with isopropanol 99%.  This should be made a few days ahead.                                                                         Working solution: 6 ml stock with 4 ml distilled water. Let stand for ~10 minutes and filter.  The filtering is very important to remove particles. But if you let this solution stand too long it reduces the staining ability. So you will have to find the best times etc.

1. Post fix  tissue in formol calcium,  Typical sections are from 6u to 10u. But you may find it helpful to cut a little thicker when you are beginning to use this stain.       2. Rinse in 60% isopropyl alcohol.                                                                                       3. Place in slides in the working solution of ORO and leave aprox 10-30 minutes.    4. Some times differentiation is need but you can also lighten the stain so be careful.  To differentiate  place in 60% isopropyl alcohol.   It is good to use the microscope at this stage to see what is happening.                                                       5. Wash in distilled water.                                                                                                         6. You can counter stain also Carrazzi  Haematoxylin.                                                   7.  Mount in glycerol gelatin (This should not be hot or you will destroy the stain).

good luck                                                                                                                         

Thank you!

C. Churukian. Really fun article!

http://www.nyhisto.com/wp-content/uploads/2011/08/Onstage_V30_I1_1210.pdf