I need to stain the neutral lipids in WAT with Oil Red O, so I have to include it in OCT but I have problem to cut the tissue with the cryostat because it stay too soft to be cut. Is there another way to fix it other than with PFA or formalin? Or another way to stain the tissue? Thanks
I found fixing in 10% formalin at 4C for 3-5 days worked well for maintaining morphology and was never successful using OCT. Perhaps there are better ways to quantify neutral lipids - an assay of some sort. Everything will be red with Oil Red O.
Yes I know that everything will be red, this is a experiment that my new lab has made before (and everything was red), but they want me to do it again...The problem is that we want to quantify cholesterol in a tissu with a large majority of triglycerides.. I just found biotinylated derivative (BCθ) of perfringolysin O (θ-toxin) which is a cholesterol marker, but I'm not sure if it is for an intra or extra cellular staining. I keep going my research... Thanks
I'm not the expert in studying cholesterol, however, I spoke with a lab who is. They routinely use GCMS for tissue analysis, however, they suggested the kits below which may require isolating adipocytes from the tissue. You can investigate further.
Both are from Abbott:
Cholesterol
07D62-20
Triglycerides
07D74-20
These solutions are for a robot, but we have used them manually with a 96-well plate. Both are colorimetric assays I have used on mouse plasma and bile. Solid tissue lipids were extracted and analyzed with GC-MS.
Good Luck,
Becky
I have had zero luck cutting frozen sections of adipose tissue. It's just not possible, they have to be paraffin-embedded. If you want to quantify cholesterol, perform a lipid extraction from the tissue and run your favorite colorimetric assay. I like the kits provided by Wako Diagnostics (Cholesterol E, 439-17501), but there are plenty of other options. I have included a link for an extraction method which I think is superior to the old Folch or Bligh and Dyer methods. Leave ORO staining for cultured cells. It's not specific for cholesterol anyway. As pointed out above, it will stain everything and answer nothing.
Article Lipid Extraction by Methyl-Tert-Butyl Ether for High-Through...
I know this is almost impossible, but I read in the instruction manual of the cryostat that the WAT must be cut at -50 ° C, what I'll try next week. We already have quantified the cholesterol, staining was to have some pictures, so if it doesn't work, is it not a big deal I think.
I have tried, as you say, to cut them at extremely low temperatures, and still was not happy with the result. If you just need a picture, a trichrome stain on paraffin-embedded sections looks nice. Even easier is a simple H&E (or really, just H). Leave the hemotoxylin on longer than normal and skip the acid step. The morphology comes out well-defined enough that I have been able to define cell boundaries automatically with imaging software.
I have used Nikon NIS-Elements in the past, although I'm sure the freely available ImageJ software from the NIH could also be used for this purpose. Any software with a thresholding function should work, as long as the borders are well defined enough. In Elements, the "Detect Edges" image function works very nicely as a starting point.
Just in case in anyone is still looking for a good way to do imaging of adipose tissue, here is a fantastic article about that (see attachment).
For cutting white adipose tissue, embedding in OCT is fine. I snap freeze the WAT immediately in -80°C isopentane. Then take a small piece from the tissue and embedd it in OCT for cryocutting. Store it at -80°C and whenever you are ready to cut the tissue, precool the cryocut to -30°C (object), -25°C (knife). Trim the tissue at 20µm and for fine cutting use 10µm. That is also perfect for Oil Red stain. I then store the sections at -80°C or directly stain for neutral lipids. For fixation I use 4 - 10% ice cold formalin for 10 min.
Let me know if you need any more help, or if I should send you the Oil Red protocol.
Hi Katrin Kaineder.
I appreciate if you could send to me the protocol
Thank you.
Hi everyone,
I am planning to see the rate of lipolysis in the adipose tissue (mice).
What is the best way to store the adipose tissue for biochemical and microscopic studies and it will be of great help if someone suggest the perfect lipolytic markers for looking at the rate of lipolysis.
Thank you.
Katrin Kaineder
Hi! Thanks for sharing!
I know if an answer from 5 years ago, but just by any chance if you still have the Oil Red O protocol? If yes, Could you please also send me a copy? I just started the work with fat tissue, it would be really helpful.
Thank you!
Katrin Kaineder
If you could send me the protocol too that would be great! Thank you in advance.
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