Hi if your lipid droplets are large, they will want to float out from the section. The old problem of "oil and water". So you need to be very gentle with the application of the staining solutions and the washes. Also I would do the staining with the slides horizontally on bars, over a sink, rather than in a rack or Coplin jar. How are you coverslipping/mountant the slides?

Our protocol: fix in 37-40% formaldehyde for 1 minute.  Rinse well in DH2O, place in ORO solution for 15 minutes, rinse well in DH2O, counterstain in Lillie's Hematoxylin for 1 to 2 minutes, rinse in DH2O, dip 2-3 times in 0.05% Lithium carbonate (bluing), rinse well in DH2O.  Coverslip from water with aqueous mounting media.

Thanks a lot Peter. I was using a Coplin jar. That sounds nice to staining slide horizontally. I will try that.

After the final wash I keep the slides 5 min to dry and then mount with glycerin.

Thanks Dolores for providing your protocol. I have never used Li2CO3. I was struggling to get the blue color as well.

Thank you.

You can "blue" with tepid tap water as long as it is not too acidic. Anything like a weak solution sodium bicarbonate will blue as it is mildly alkaline.

Try using Kaisers for coverslipping, it is a aqueous "setting" mountant. You can get a nuclear artefact if you dry the tissue before mounting.. Basically it is trapped air...

I will dig out my Oil Red O protocol for you.

Hello Peter,

Thanks a lot. It would be great to have the protocol. Next week I will try another set of slides.

http://www.ihcworld.com/_protocols/special_stains/oil_red_o_ellis.htm

we use the above technique. I find that the 60% isopropanol washes, remove the precipitation problem of ORO on to the slide and avoids false positives. We stain for 30 seconds - 1 min using Gill's H. And blue in tepid tap water, coverslip with Kaiser's mountant, ring the coverslip if need be with nail varnish. It takes an age to filter the ORO so do that at the start of the day..

Thank You so much Peter..!