oil red o on thicker sections

[macaronee]

Hello,

I have been using the Oil Red O protocol from this forum. My experiment consists of staining ex vivo human fat from the abdomen. I embed the fat in OCT and section at 50ums (that's where I can have nice frozen sections that dont melt and/or crumple).

I realize that the protocol calls for 5-10um thick (how do you guys section fat this thin?!). I still tried to see if it will work and even increased incubation time since my sections are thicker. However, my stains look very spotty. The red stains look "spilled out" from the cells. These are supposed to be untreated fat samples so I was expecting intact cell membranes and the lipids to stay "inside"

Any suggestions on how to fix this?

Please and thank you!

-Melissa

[excalibur]

Cutting frozen sections of just adipose is nearly impossible. Oil Red O is usually used to locate fat in other tissue such as liver or a fatty embolism in lung.

For studying just adipose tissue I suggest you fix with 10% NBF and then post-fix with osmium. Osmium fixes the fat in such a way that it can even be processed to paraffin.

http://stainsfile.info/StainsFile/prepare/fix/agents/osmium_tetroxide.htm

[MT Scientist]

Sectioning will also disrupt some cell membranes causing the fat to "spill out".  You may also have enough water present in your sample to create ice crystals resulting in membrane holes.

Sounds like a "smear" preparation might work for you.  However, why you are sectioning fat is not clear from your post.....

[macaronee]

Thank you for the responses.

I am sectioning fat to see difference between heat-treated and untreated fat. I do have to section frozen adipose tissue (and have successfully done it, but it has to be 50 um thick).

I would really like to be able to stain the lipids confined within the fat cell, especially for the untreated fat block. For now, I am going to try to fix the tissue first in either 10% formalin or 4% PFA then cryoprotect in 30% sucrose before embedding to see if that will prevent disruption of cell membranes.

I would eventually try to use this protocol for staining/locating lipid in other tissue (which would be cut frozen about 30um thick). For now, I'm trying to optimize

Thanks again!