[Histonet] Oil Red O Churukian method, reposted

[Gayle Callis]

Dear All who want this Oil Red O method, the none messy type! 

Enjoy

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)


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Oil Red O/Dextrin, Churukian method

Fresh tissue (not prefixed) frozen sections are immersed immediately into
Neutral buffered formalin for a minimum of 10 min (can be hours/days).
Rinse sections with distilled water before staining.  Prefixed, with NBF
should be cryoprotected in 20 - 30% sucrose,  mounted on Plus Charge slides
and air dried for 30 min to 1 hour, or longer to insure sections stay on
slide. Do not fix frozen sections with alcohol or acetone to prevent lipid
removal. 
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 Scotts tap water type 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 and 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.
Reference:  Gamble and Bancroft, Theory and Practice of Histological
Techniques, 5th Edition, 2001 with photos.  This method also published in J
of Histotechnology by Charles Churukian.  Go to JOH archives for journal
year/volume.