Ada Feldman sent me an article on Oil Red O staining and I wrote the following protocol for my lab based on the article. It works exceptionally well, frozen adrenal controls have brightly stained positive cortex and clear negative medulla. Best of all it has no precipitate like all the other methods. It also birefringent under polarizing filters. This is the best Oil Red O protocol anywhere!
Our lab has tried several water based mounting mediums and the warm glycerin jelly works best. With glycerin jelly the stain and hematoxylin fades very little, even after a week. Also the cover glass doesnt slide around because the jelly firms up when it cools. OIL RED O IN TRIETHYL PHOSPHATE From: Ada T. Feldman and Richard W. Dapson (1974) Medical Laboratory Technology 31, 335-341, RELATIVE EFFECTIVENESS OF VARIOUS SOLVENTS FOR OIL RED O, Department of Biology, University of Michigan-Flint, Flint, Michigan 48503, U.S.A. Triethyl Phosphate (Spectrum T2256, 500ML, CAS 78-40-0) VWR cat. # 700006-688 Oil Red O (Alfa Aesar CAS# 1320-06-5, 25G, C.I. 26125) VWR cat # AAA12989-14 Glycerin Jelly Mounting Medium (Electron Microscopy Sciences, Cat. # 17998-10, 100 ml) VWR cat# 17998-10 VWR Harris Hematoxylin (VWR Premium Stains, 95057-858) VWR cat # 95057-858 Lithium Carbonate (TCI America, 500 G) VWR cat TCL0224-500G Disposable Transfer pipettes VWR cat# 16001-180 VWR Superfrost Plus Microscope slides (VWR 25X75MM PK72) VWR cat# 48311-703 VWR® Micro Cover Glasses, Rectangular, No. 11/2 VWR cat# 48393-194 Solutions: 60% Triethyl Phosphate 600 ml Trithyl Phosphate 400 ml Distilled Water 0.5% Oil Red O in 60% Triethyl Phosphate 300 ml 60% Triethyl phosphate 1.5 gm Oil Red O Harris Hematoxylin Saturated Lithium Carbonate 18 gm lithium carbonate 1500 ml Distilled Water Technic: 1. Pre-warm Glycerin Jelly in 60°C waterbath 30mins prior to staining. 2. Bring frozen sections to room temperature. 3. 60% Triethyl Phosphate a few dips. 4. Stain sections in 0.5% Oil Red O, 15 to 20 minutes. 5. Rinse in water 2 minutes. 6. Counterstain in filtered Harris Hematoxylin, 2 minutes. 7. Blue in saturated Lithium Carbonate solution, 10 seconds 8. Rinse in water 5 minutes and hold in water. 9. Mount with warm Glycerin Jelly using a clean transfer pipette. Take care to avoid contaminating the pipette and Glycerin Jelly. Results: Fat Pink, Red, Bright Orange Nuclei - Blue Donna J. Emge, ASCP-HT Mouse Histology and Phenotyping Laboratory Manager Northwestern University Olson Pavilion 8-333 710 North Fairbanks Court Chicago, IL 60611 d-e...@northwestern.edu _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet