Alternative embedding for histology

[Naphthalene]

Hello!
I want make a little communication to share my thoughts on embedding of samples for histological processing.

For home histology, the paraffin processing appears the most attractive option (when compared to frozen and plastic embedded sections, which require sophisticated techniques and advanced equipment for their successful use). However there are some limitations of paraffin technique: the necessity of complete dehydration and extraction of lipid (fat) components of all cells for effective paraffin infiltration of tissue, that leads to tissue shrinkage and impossibility to study the lipid cell components. During floating of sections on water bath the tissue expands. However, since the tissue remains paraffinized during flotation procedure, it only expands to the size of paraffin embedded tissue, that can be seen on the cut surface of paraffin block.
Even after lipid extraction, the fixed dehydrated tissue preserves its ability to revert to its initial volume upon rehydration. The paraffin sections are traditionally mounted on slides after their stratching on water, and after being affixed to the slides they loose their ability to expand further to their initial volume upon rehydration.

That limitations of tisssue expansion should not be expected if the sample is embedded in water soluble polymer. If tissuse section, that is embedded in water soluble polymer is floated on the water bath, the polymer gets dissolved. The tissue section stretches completely as far as it wants, and can revert to its initial size without experiencing the influence/limitations of embedding medium, that is no more present in the tissue.

My own experiments with brain tissue revealed (confirmed the already known facts) that the soft tissue is strongly shrinked when its is processed through a graded series of alcohols, to displace the water with alcohol. At the same time, if the same tissue sample is processed through the inverse order of alcohol dilutions to water, it restores completely to its initial volume. That is observed, even despite the loss of fat/lipid components that were extracted during alcohol dehydration procedure!

Polyethylene glycol with molecular weights of 1000 fulfills all above requirements: it is water soluble, and it can be cut easily at fine thickness. PEG 1000 is especially attractive because it melts at 37C that is much lower than paraffin. It allows to preserve the the enzymatic activity of many proteins upon tissue embedding, and it does not extract the lipids because PEG is very polar/hydrophilic. Though PEG is much less popular than paraffin, it has many perspectives as an embedding medium for histology.

The disadvantage of PEG is its hydrophobicity that leads to difficult handling of sections.

For inspiration I want to show you how PEG 1000 can be cut at 6 micron thikness (it can only be done at cold dry conditions since it is very hydrophobic and sections merely dissolve on the knife if humidity is too high).

[Raul]

Hi Naphthalene, very interesting facts here. Could you please show some pictures under the microscope comparing the quality of the tissue with PEG compared to paraffin embedding?

[Naphthalene]

Hello, Raul!
I have only started to study that PEG embedding method, and I haven't made any mounted sections yet. I have made several attampts to embed and cut the tissue: however it was revealed that all steps of the process require proper technique.
The two main problems that I have encountered were the bubbles and cracks in the PEG blocks, and the handling of the sections themself. The first problem was solved by slow casting of the blocks, by leaving them to solidify at room temp without any external cooling. The second problem is more severe, as the sections instantly melt upon contact with fingers (gloves might help) or even moisture from breath, just like the frozen sections, so this step requires additional training.
I'll post the images of sections as soon as I will succeed in their making.

[Peter]

Hi Naphthalene,
Do the blocks of PEG readily form ribbons of sections?
Peter,

[Naphthalene]

Yes Peter, PEG (mol weight 1000 or its mixtures with a bit of higher weight PEGSs like 3500-4000) is easily ribboned at various thickness. The hardest mixtures can be cut at 4 micron an even thinner (which I never tried). The PEG1000 compression is adequate when you cut ribbons at 8-12 microns, but handlling of PEG ribbons would be quite difficult. On my pictures above you can see such ribbon of PEG sections.

It should be mentioned, that since the PEG ribbon will disappear after placing it on water, the sections will start swirling on the surface and the initial order of sections will be lost. To carefully preserve their order as they came from microrome blade it is advantageous to mount the sections one by one. The technique and the properties of PEG sections seem to resemble more that of frozen, than paraffin sections.