[Histonet] non messy Oil Red O staining

[Gayle Callis]

This method developed by Chuck Churukian is less messy than the oil red O stain using propylene glycol.   We have used it for fresh tissue frozen sections, fixed in NBF OR in formalin fixed tissue, snap frozen, cut and then stained.  Chuck published this in J of Histotechnology and it also is found in Gamble and Bancrofts 5th and 6th editions of Theory and Practice of Histological Technique.  You microbiology department may have the correct dextrin available.  

Oil Red O/Dextrin, Churukian method

 

Frozen sections fixed post cutting with NBF can be rinsed and stained immediately, or NBF fixed tissue 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.  Fresh tissue frozen sections can be fixed 10 minutes or so before staining, but treat these gently. 

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.



Reference:    C. Churukian, Lillie's Oil Red O for neutral lipids, J Histotechnology 22(4):309, 1999.



Gayle M. Callis

HTL/HT/MT(ASCP)

Bozeman MT 59715