Re: [Histonet] [Fwd: urgent H&E issues]

From:Bryan Llewellyn

When I used to do this with 1 micron sections of kidney in methyl/butyl 
methacrylate I would cut the section with a 20% acetone filled trough 
attached.  I picked up and floated onto a water bath kept at 65  C, lined 
several section up and mounted on the slide.  I baked on for at least a 
couple of hours.

To stain, I removed the plastic with an hour in acetone, xylene or, 
preferably, chloroform at room temperature.  I used toluidine blue for the 
stain.  H&E came out very pale, even if I used Cole's hemalum for an hour at 
60 C with no differentiation.

I found that sections thicker than 1 micron were increasingly more difficult 
to deal with as the section thickness increased.  3 microns was the 
practical limit unless I was willing to spend a lot of time fiddling around. 
However, the whole point of such sections was to cut them thinner than I 
could from a paraffin block, which I also had from a second biopsy (and a 
third for EM)..

Bryan Llewellyn

----- Original Message ----- 
From: "Esther Peters" 
Sent: Tuesday, March 11, 2008 8:31 AM
Subject: [Histonet] [Fwd: urgent H&E issues]

> I'm passing this question along from a research colleague, because I have 
> never worked with plastic (I'm not sure whether she is using MMA or GMA, 
> either).  I tried a Google search but did not see a direct answer. We look 
> forward to your assistance!
> We have been cutting some plastic here over the last few days (with a
> borrowed microtome), and are having some difficulties with the H&E
> staining. It seems that the section gets tons of little bubbles under
> the plastic (or so it seems). We do not see such bubbles when we stain
> with basic fuchsin and think maybe its due to the alcohol or xylenes used
> at the end of the H&E. We are not removing the plastic (methacrylate).
> Do you have any ideas or can track down an H&E staining procedure
> especially for plastic?
> Esther Peters, Ph.D.
> George Mason University
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