Re: By which method do Xylene's clear lipids from fixed tissue sections?

1. Ethanol is used to dehydrate because water in the tissues will dissolve/partition into the ethanol but lots of other things in the tissue (proteins for example) will not. Moreover, ethanol is sufficiently like water that replacing water with ethanol minimizes physical/structural changes in the tissues being examined. In other words, if you remove water without replacing it, a prune turns into a plum. If you replace the water with something, it will still at least be the size of the plum and look more like the plum, though I doubt it would taste great.

2. Xylene(s) is very similar to benzene and toluene. They can dissolve many lipids. They are also compatible with ethanol. If you tried the opposite order, xylenes then ethanol, the xylenes would not mix with the water, would not dehydrate and would damage the tissue.

3. Many lipids, though not all, are soluble in xylene(s) because xylene is hydrophobic ("hates water", similar to trying to mix olive oil and water for a vinaigrette for salad dressing). Lipids are also hydrophobic. They are a "fatty acid", a long hydrocarbon chain with a carboxyl group at the end. The hydrocarbon chain makes them relatively insoluble in water. Although one end of the lipid is charged and therefore will be somewhat hydrophilic (loves water), the long hydrocarbon chain predominates, so the lipid isn't very soluble in water (or ethanol) but is much more soluble in xylenes.

4. The xylenes don't remove all lipids, just a lot of lipids. Sometimes acetone is used also. Basically the purposes of the washes is to remove all the water and much of the lipid and dessicate or dry the tissue section so that the proteins and other components simply stay in place. They don't actually precipitate out, but the effect is the same.

5. If you want and interesting experiment to test out things like this, try the following. Put an egg into a plastic container. Cover with dilute vinegar (1 part vinegar, 4 parts water). Cover and leave for a couple of days. Gently rinse and look at the egg. It's roughly the same size, but the vinegar has dissolved the shell and denatured the proteins so that you get a fairly rubbery egg but still the same size egg. Then take a second egg and cover with 1 part vinegar, 1 or 2 parts water and 2 or 3 parts Karo syrup (clear is best). Again wait two days. The shell has now dissolved, but all the water has been drawn out of the egg. The vinegar has denatured/precipitated the proteins and you now have a rather small "fried" egg. It's exactly the same as the first egg, but the water is removed. The Karo syrup is removing water simply because it's a very large amount of sugar dissolved in a small amount of water. The water in the egg is simply "dissolving" into the syrup and diluting it. (The sugar is also going the other way so you end up with a vinegary, extremely sweet, very strange looking fried egg, not that I've ever tasted one, though a couple of first graders tried one time. They washed their mouths out rather quickly.)

6. I've never tried this with alcohol (cheap vodka if you don't want to swipe some lab ehtanol) or xylenes, but it might be interesting to do so. I would expect the alcohol (plus some vinegar) to look a lot like the egg with just water and vinegar though probably somewhat smaller. Don't know what I'd expect if you just used xylenes alone (wouldn't work with xylenes plus vinegar because the vinegar wouldn't dissolve in the xylenes. If you tried ethanol plus vinegar first and then tried xylenes, I'd expect the egg to get a bit smaller, but not shrink dramatically like the egg with the Karo syrup.