Hello, I tried to dye isolated lipid droplets using ORO solution (0.5%isopropanol) from Sigma and what I obtained is that the colour of Lipid droplets (from adipose tissue) was darker red (than usually obtained) and many of them were covered with black spots all arround making hard to distinguish their colour. Anyone has any idea what it may have happened?
thanks a lot
I have done it! even after flitering it with a filter syringe, I get the same spots!
I will try washing with PBS after embedding in PFA 4% to see if it gets better
thanks anyway!
Do you stain in a coplin jar or horizontally on a staining bridge? It is better to take a closed jar because evaporation during staining can lead to precipitats nevertheless of filtering before.
It can be a problem if you don't take the images right after the staining, because ORO oxidizes and precipitate again. Leaving the samples in the fridge helps.
Dears, I hope someone still gets questions from this thread.
I am wondering how to fix the cells teh right way :
PFA 4% methanol free is enough to - if let on the cells long enough, i.e. 30min - permeabilize them?
Or do I need Formalin if not using a detergent ???
I thought PFA and also Formalin alone do not permeabilize, but some people fix for 30 min (in PFA) and do not indicate any detergent but stain right after. I also know that many people just use PFA and Formalin like synonyms and might have indicated wrong....
any help is highly appreciated !!!