Re: Seeking H&E stains procedure for Ground Plastic section

From:Bryan Llewellyn

Hi Leon,
I stress that I used 1 micrometre sections.  I have never cut methacrylate
sections thicker than 2 micrometres, so my experience is based on different
parameters than yours or Bernards.

I tried several solvents for deplasticising.  Quite a few worked.  Many had
strong smells, or reputations of toxicity.  I settled on acetone for one
hour since it worked and I didn't mind the smell.  It appeared to remove all
of the methacrylate.  When I was first learning how to do this work I did
get lots of sections coming off the slides and lots more buckling (small
areas lifting off but most areas attached).

I tried lots of adhesives, but most didn't work.  The best was burnt
albumin, a rather obscure procedure (given at end), but it wasn't good
enough either.  I finally increased the temperature of my water bath to
about 70C, picked up the sections from 20% acetone and floated out.  I
picked up on freshly made, absolute ethanol soaked, individually polished
slides as oil free as I could make them, and baked on at close to 70C for at
least two hours, sometimes overnight.  I had almost no sections lifting when
I did this.

The hour or so in hemalum was to get as much dye into the nucleus as
possible.  They were so thin that normal staining was far too pale.
Increasing the time and temperature made a significant difference.  Since
there was no methacrylate left, there was no particular resistance to dye
penetration.  This is why I stress the thickness.  Both yours and Bernard's
may still have plastic present.

To use burnt albumin, smear Mayer's albumin THINLY on a slide.  Hold it in a
flame until it smokes.  Cool, pick up the section and don't reorient.  Bake
on well.

There is another adhesive I have used which stuck sections to glass
incredibly.  It is of no use for regular work since it is a fiddly and
obtrusive, but it is better than nothing.  It is (in Canada) Silicon Seal.
Try the original uncoloured acetic acid smelling stuff.  Smear a small
quantity on a slide and pick up.  Bake as usual until it is well cured.  It
worked for me with sections of 10 micrometres (not plastic) for a technique
in protein histochemistry.  I remember that it was used as the mounting
medium as well.

Bryan Llewellyn

----- Original Message -----
From: "Driessen, L." 
Sent: Monday, January 06, 2003 1:15 AM
Subject: Re: Seeking H&E stains procedure for Ground Plastic section

> Hello Bernard,
> I'm working on a simular problem with my methyl methacrylate saw-sections.
> Normally I remove the methyl methacrylate out of cutting-sections (7
micron thick) with 3 steps of chloroform/xyleen 1:1 (15 minutes each), 15
minutes xyleen and then bring my slices through alcohol to water. After that
I can perform a normal H&E-stain.
> I've also tried to use this method on my saw-sections (20-40 micro thick),
but then my slices come loose from the glass-carrier when I leave them to
long in the chloroform/xyleen mixture (before sawing I fix the block to a
glass-carrier with UV-curing glue).
> In my case it only works with a short deplastication-time of about 5
minutes. Maybe this is fine to you.
> But now I also have a question for you. Are you familiar with my problem
of slices coming loose, and if so, how do you pervent this (I expect you
attach your slices to a glasscarrier)?
> Greetings Leon Driessen
> P.S. I would like to ask the same question to Bryan Llewellyn.

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