[Histonet] Re: Polyester wax -it does work for me too

[Stanislav Vitha]

Hi Rebecca,

I just joined the mailing list and found this old thread on Steedman's wax. 
I am glad Steedman's wax is working for you. I have used it extensively in 
last several years with good results for immunofluorescence labeling of 
many different epitopes in plant tissues (expanded leaves, meristems, root 
tips, flower buds, etc (I also embedded and sectioned mouse embryos and it 
worked fine as well).  Using a standard, old-fashioned AO/Spencer rotary 
microtome, I am routinely able to get 5 micron sections if the 
air-conditioning works reasonably well, no need to use cold room or chill 
the block or the knife in the refrigerator (at least for the tissues I have 
worked with; it may help if you want even thinner sections).  My specimens 
are usually rather small, the face of the sectioned block is generally 
about 5 x 4  mm, so for larger blocks it may be not as easy. I did not have 
the Section Transfer Station on my microtome, but several tricks helped:

1) During sectioning, I use a fine paint brush to lift the end of the 
ribbon and to hold the ribbon in the air so that it does not slide on the 
knife (it tends to stick to it if you do not)

2) For the 5 to 10 micron sections I use high sectioning speed (turn the 
wheel as fast as you can) and produce  a ribbon up to about 30 cm long. I 
have cut 20 micron sections occasionally, using slower cutting speeds, but 
they tend to curl. I then use a second paint brush to lift and dislodge the 
other end of the ribbon from the knife edge.

3) I place the ribbon on a wooden board or a sheet of paper and using a 
razor blade, cut the ribbon into smaller pieces and place them on a dry, 
poly-L-lysine coated slide. - if you tilt the razor blade when cutting the 
piece, the short ribbon will stick to the edge well enough so that you can 
lift it and lay it on the microscope slide.

4) Add a drop of water to one end of each ribbon on the slide. The water 
will run under the ribbon and it will expand and stretch. Wick off excess 
water with filter paper.

5) Allow to dry at room temperature for few hours or overnight, then 
proceed with dewaxing and staining/immunostaining.

More details and pictures on embedding and sectioning  can be found in the 
following book chapter:

Vitha, S., Baluška, F., Jasik, J., Volkmann, D., and Barlow, P., Steedman's 
wax for F-actin visualization, in Actin: a Dynamic Framework for Multiple 
Plant Cell Functions, C.J. Staiger, F. Baluška, D. Volkmann, and P. Barlow, 
Editors. 2000, Kluwer: Dordrecht, The Netherlands. p. 619-636.

You can download the PDF from 
http://www.izmb.de/volkmann/pdfs/  (Book-Steedman's_wax.pdf)

The disadvantage of the above method is that the ribbons sometimes do not 
expand
100%, or al least not all sections; for what I was doing it was not 
critical, I was more interested in cellular and subcellular immunostaining 
rather that looking at tissue and whole-organ morphology.

For some of the immunolabeling, I have also used Superfrost Plus slides, 
but the section adherence is much worse than with the poly-L-lysine slides, 
especially if one needs to do antigen retrieval and/or lengthy incubations.
(For some epitopes, I had to autoclave the tissue in high-pH buffer, and 
all sections were lost from the Superfrost Plus slides, while most remained 
on the lysin-coated ones).


Good luck!

Stan Vitha


>From: 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"  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?



Dr. Stanislav Vitha      vitha <@t> mic.tamu.edu
Microscopy and Imaging Center
Texas A&M University
BSBW 119
College Station, TX 77843-2257

tel: 979-845-1129 (main desk)
tel: 979-845-1607 (direct link)
fax: 979-847-8933