[Histonet] Oil Red O Stain on Fluid

[Gayle Callis]

You cannot fix cells containing fat/lipids with alcohol or acetone or the
lipids are removed by these solvents. For cytospins and cell cultures - fix
with NBF or paraformaldehyde, rinse and do the Churukian Oil Red O stain,
coverslip with aqueous mounting media to retain Oil Red O staining. 

Oil Red O/Dextrin, Churukian method:
Fresh tissue frozen sections fixed post cutting with NBF can be rinsed and
stained immediately, or fixed frozen sections (cryoprotected), mounted on
Plus Charge slides. Air dry NBF fixed frozen sections 30 min to 1 hour, or
longer to insure they stay on slide. Cells can be cytospun, cultured, and
fixed with NBF.  
Protocol:
1.	Immerse dry slides directly into filtered 0.5% Oil Red O in Dextrin ,
stain 20 minutes
2.	Rinse VERY GENTLY in running tap water
3.	Counterstain with Gill II hematoxylin for 20 - 30 seconds
4.	Rinse gently with water, blue in bluing solution, (NOT AMMONIA WATER),
rinse gently, and coverslip with aqueous mounting media
Reagents:
Dissolve 0.5 gm Oil Red O in absolute isopropyl alcohol, allow to stir
overnight.
Dissolve 1 gm dextrin (bacteriological grade or TYPE III (Sigma) from corn
in 100 ml distilled water
Working solution is 60 mls stock Oil Red O and 40 ml 1% dextrin solution
Stable for months, and reported to work on paraffin sections.

This is a very clean stain, and not MESSY!


Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)