[Histonet] Re: Polyester wax (with earlier citations)-it does work

[Rebecca Nishi]

I really appreciate everyones comments. It sounds like most everyone is not
that excited about it.

I too read all of the literature from the 50s onward (there isnt too much),
and have tried Steedmans, and PEG-Disterate from Sigma. The procedure isnt
really too bad.

As for my results so far, they are actually pretty good. I was hoping to get
some help with the fine tuning.  I am confident I can get good 10 um
sections, using a rotary microtome (from Microm), I tried it with and
without a cool-cut adapter to keep the specimen cold (I think it was around
-10 to +4 C, I forgot), cold didn¹t seem to help so we didn¹t get that. But
what  helped tremendously was the STS (Section transfer station). It is a
little waterfall and water bath, set to 30C. The sections slide perfectly
and flat down the waterfall, and into the bath, and are immediately mounted
on Superfrost Plus slides. I dry them on the slides for a day or 2 at room
temperature or 4C, then bake them at 30C -40C for about 15-30 minutes.

I think a cooler room definitely helps. But I was cutting on the rotary
microtome, here in Southern California (mostly sunny, ~70-80F at the time)
and had no trouble when using the STS. We were having a lot of trouble
cryostatting recently (The past year), and thought we could get more stable
sections with this wax. I tried cutting the wax on the cryostat and with a
sliding microtome with little success.

I was able to get nice 20uM sections as well, but with my current adhering
protocol, they came off the slides during staining. I plan to work on that
soon.

I was unable to get 30 uM sections, they crumbled when hitting the knife. I
think 10 and thinner is optimal, but I would like to get 20 if possible.

I can easily get ribbons that float on the pool. Once the section or ribbon
goes under the water, they are a little bit hard to handle, and unfold. You
have to put on a slide right away. Also, I don¹t think you can store the
ribbons like other types of wax.  I am only going to put 1 or 2 sections per
slide anyways because I plan to do stereology, and want multiple sets for
various stains.

I think this method does have some potential, and I am particularly
interested in using this for injured areas, which tend to fall apart with
cryostat. I really have not found it that difficult to use, so far.

Rebecca

On 5/4/04 10:23 PM, "John Kiernan" <jkiernan <@t> uwo.ca> wrote:

> We tried Steedman's polyester wax in the early 1980s (Yes,
> after reading the early 1950s literature!). Powder rather
> than sections poured over the knife's edge. We gave up.
> 
> In the light of recent (1990s) advances, are there any
> reasons for trying to master the lost art of sectioning
> polyester wax? This embedding medium was introduced
> shortly before the cryostat and long before antigen
> retrieval. In one of Steedman's procedures polyester wax
> was included in a one-step mixture with a Bouin-like
> fixative.
> After 24 hrs the liquid was cooled, and when it had set
> you could trim the block and (he said) produce ribbons
> of sections.
> 
> Polyester wax reappeared in the 1970s, when it was widely
> thought that immunohistochemistry worked best after little
> or no fixation and avoidance of organic solvents, wax,
> heat etc. This didn't catch on despite being published in
> classy journals. 
> 
> The comments from Stephen.Eyres <@t> sanofi-synthelabo.com
> (cited below) should help those who try to cut polyester
> wax. Clearly it's a difficult medium to handle. Are there
> any purposes for which it must be used?