Dewaxing

[TonyT]

I finally managed to get a half-decent cross-section of a 1-year-old eastern White Pine (Pinus strobus) twig using my recently acquired Lipshaw sledge microtome
viewtopic.php?f=14&t=14360
It does not have a correct knife, needs one 220 mm long (hard to find!), but I did manage to clamp a knife to the microtome.
I am still having problems with wax embedding, not getting complete penetration, resulting in a less-than-complete section and having to make thick sections.
The reason for this post is to show that "Goo Gone" can be used to remove the paraffin wax from a section. I added a drop of the liquid to the section, on a slide, and heated it to 60 C; washed the slide in 99% IPA. Finally stained in Safranin-O and Fast Green.
I have been using Xylene to dewax in the garage in freezing temperatures (this is Canada). Using "Goo Gone" indoors is a real bonus.
Olympus DPlan 4x + 1.25x + 2.5x, 30µ section; polarized

[Microscopy_is_fun]

Hi Tony,

the active components in GooGone which dissolve the wax are limonene:

https://en.wikipedia.org/wiki/Limonene

these are quite commonly used as replacement for xylene in histology:
https://www.carlroth.com/de/de/xylol-er ... l/p/6640.4

Although less toxic than xylene, they are still hazardous. Just look at the hazard classification in the linked Wikipedia article.

[MikeBradley]

I hoping to use d-Limonene to dilute my stock of Brunel Numount which has become too viscous to use, even when sitting on a coffee cup warmer. As amateur microscopists in Canada, we find it very hard or even impossible to source many useful products by regular mail

Michael

[TonyT]

The GooGone was soluble in 99% IsoPropyAlcohol (Shoppers Drug), so maybe it will liquefy your mountant

[MicroBob]

Hi Tony,
for paraffin embedding I have used this method: https://www.klaus-henkel.de/isomethode.pdf
I do infiltration and de-paraffinisation in small closed containers so there is no intense smell, even using xylene.
Infiltration twigs should not be a big problem as the cells are fairly open.

Bob

[TonyT]

Thanks Bob
I made a similar set up but using small vials; working with the xylene in the garage.
Did manage to get a reasonably complete cross-section of 1yr-old white pine (Pinus strobus) @ 20µ using an old knife.
I have bought a large Lipshaw knife that will fit the microtome; should be here after Christmas.
In think one problem is air in the vessels of the samples; do people use a vacuum pump?
Brightfield, safranin + aniline blue; Euparal mount.

[BramHuntingNematodes]

I like Gray's suggestion of using heat from above, from an incandescent light bulb, to melt the.wax. careful positioning so that the wax melts halfway ensures that samples introduced will suffer the least amount of damage from heat.

I have thought about a vacuum chamber, particularly when using caustic soda to soften specimens. I haven't tried it yet and so have substituted time instead. I leave stems in molten wax for as.long as I can bear, maybe a day or so. I will see if this helps as soon as get my 'tome running again.

[MicroBob]

Hi Tony,
nice results!
The tear in the middle of your last section seems to appear when the knife passes the marrow. The xyleme is then torn open a little. It is fairly hard material, probably even more so after the fixation and dehydration, so th esupport by soft paraffin may be on the low side. It might help to cool the block before sectioning and cool the surface by holding a cooled object slightly above it, I use a car valve shim for this, steel disc 4mm thick.
With your new knife you can make more of a slicing cut, this will help too.
Vaccuum will help, especially when applied and released many times. It might also help to place the infiltration bath in an weak ultrasonic cleaner for a moment.

My next experiments will be to test Technovit 7100 resin for embedding.

Bob