Oil red O formalin fixed tissues

From:Gayle Callis

Formalin fixed tissues are cryoprotected with sucrose solution, 15 - 20%
overnight in refrig, tissue sinks to bottom of vial, blotted, embedded into
OCT and snap frozen.  Sections are cut, mounted on plus charged slides and
allowed to dry at RT for 30  minutes - 1 hour, we use the Churukian Oil Red
O procedure, very clean, no messy propylene glycol method.  I have frozen
FF tissues after rinsing, but they never cut as nicely as cryoprotected,
always crunchy with too much ice. Have blotted excess water out, and it
works, but not as well.  I must admit, fresh tissue snap frozen give
wonderful ORO with this method, and superior to FF tissue.  

I don't even bother to rinse OCT off although if you do that, but rinse
gently, stain in Churukian ORO solution for prescribed time in method,
rinse in running tap water, counterstain with Gill 2 hematoxylin to desired
depth of color, usually 15 seconds or longer, rinse, blue, rinse with tap
water and mount with Aquamount.  Never press on the coverslip as you can
push the stained lipid (displace it mechanically), let it dry and view!  

I have heard some people had trouble with CrystalMount, but never use this
except for very special IHC protocols.  Could be there is an
incompatibility of this mounting media with the Oil Red O stained lipid
(Oil Red O is soluble in lipid droplets) or is it vice versa??  If you push
on coverglass to move it around or squeege out excess mounting media, I
think you displace things, even create bubbles. Been there, done that! 

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu

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