[Histonet] Re: Etching plastic for T blue staining
gayle.callis <@t> bresnan.net
Mon Jun 8 13:19:43 CDT 2009
Chris, If you do not do this, consider etching your tissue to remove the
surface plastic. We use 0.2% formic acid - start with less than a minute as
your tissues are thin. Some labs etch using ethyl alcohol or methanol. We
Using the formic acid is commonly done on undecalcified bone embedded in
methyl methacrylate to achieve a mild surface decalcification of the bone.
This removes a few micrometers of calcium from bone in thicker slab section
(ground and polished) and on thinner microtomed MMA sections. However, I
don't think the acid actually dissolves/removes plastic itself. This is why
acid etched plastic bone permit certain low molecular weight dyes (toludine
blue, basic fuchsin, methylene blue and dye mixtures e.g. Sanderson's rapid
bone stain and Shenks recipe MacNeals tertrachrome) to penetrate into the
bone matrix of thicker slabs.
However, etching the plastic with alcohols is a good way to soften and
possibly remove some of the plastic to allow better penetration of dyes into
the soft tissues. Glycol methacrylate is not removable, MMA is removable
using xylene and some other solvents, and if you use an EM resin, you may
have to do sodium ethoxide treatment to remove plastic. For EM resin
embedded sections at 1 um or so, we uses Toluidine blue/sodium borate , pH
11, by flooding a section then heating on a hot plate, rinsing, drying and
coverslippling. Most of the time, tissues embedded in GMA or MMA will
stain with a toluidine blue method, particularly when the pH is 8 or higher.
There is a very good discussion on the etching, dyes, pH and temperature
effects on various plastics in this publication. Horobin RW. Staining
plastic sections: a review of problems, explanations, and possible
solutions. J Microscopy 131:173-186, 1982.
Gayle M. Callis
Bozeman MT 59715
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