I would like to cut the adipose tissue using a frozen section (10 μm) and do an oil red o staining.
I have never done this before so I do not know how. Everyone seems to have a different way.
The cells are stained with oil red o, but the method of tissue formation is not well documented.
Can anyone tell me about the protocol?
Thank you.
Hi Jinju,
this is our protocol: the dye Oil Red O is in the form of powder, to be dissolved in isopropanol (0.5% w/v); afterwards, it must be further diluted with deionized water, in a ratio of 3: 2, and filtered. For staining, after PBS re-hydrtaing step, the sections are incubated for 40-45 minutes with reconstituted Oil Red O, at room temperature. They were rinsed with PBS and lmounted to be observed at the microscope.
Good luck
It helps to heat the iso propanol up when making the saturated stock Oil red oil. Heat it safely, in a Bain Marie. Do a negative stained slide as well. As it is possible to get false positive staining if your staining technique is not good. Use acetone to remove the lipid from a parallel section and all that should stain up is the nuclei with the H. That way you ensure your staining is genuine. And Kaisers is a good setting aqueous mountant.
You need: 1. 100% propylene glycol
2. 0.5% oil red o solution(0.5 g oil red o+ 100 ml Propylen glycol 100%)
3. 85% propylen glycol solution(85 ml propylen glycol 100% +15 ml dist.water)
4.Mayer's hematoxylin solution
5/Glyceri jelly
Protokol:
1.rinse your frozen sections in DW(distilled water).
2.2 min in 100%propylene glycol
3. 16 hours in oil red O solution
4. 1--3 min in 85% prop.glycol solution
5.rinse twice in DW
6.1 min in Mayers'hematoxylin
7.twice in DW and mount in warmed jelly
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