Re: MGP
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | Jim Manavis <jim.manavis@imvs.sa.gov.au> |
Reply-To: | |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
On Tue, 21 Mar 2000, Jim Manavis wrote:
> Can anyone help with the technique MGP (Methyl green pyronin). It assists in
> differentiation between DNA and RNA. One of the pathologists has asked for
> it.
No replies have appeared on the HistoNet listserver yet, so here's quite
a long one to give you some encouragement.
First-off, the method should no longer be called MGP. It should be
EGP because nowadays the DNA stain is not methyl green but ethyl
green.
This is a _superb_ staining method. It was splendid even in the old
days, but it became technically easier with the advent of dyes that
were pure enough to behave reliably. This happened in the 1970s but
knowledge about the better dyes was slow to spread, and it's only
in the last decade or so that the technique has shed some of its
reputation for being troublesome, difficult, fickle etc.
The important thing is not to use dyes that have been hanging round
on some shelf for more than 20 years. It's not that they change with
keeping. The newer ones with the same names on the labels have higher
dye contents and don't contain undesirable impurities.
There are many variants of the technique. With modern dyes and a
suitable procedure the only skill that's needed is in avoiding
undue loss of colours (greenish DNA, red RNA, orange metachromasia
of mast cell granules & cartilage). It's generally agreed (I think)
that water extracts the green component and that water-alcohol
mixtures, ethanol and acetone extract too much of both dyes.
n-butanol is the dehydrant of choice, after thoroughly shaking and
blotting the slides to remove most of the dye solution.
Use a procedure taken from a book or paper published less than 10
years ago. If the instructions tell you to purify your methyl green
solution by chloroform extraction, they are out of date. True methyl
green (CI 42585) always needed this treatment, but it hasn't been
manufactured for many years. The dye sold under this name is really
ethyl green (CI 42590), and it doesn't contain crystal violet as an
inevitable chloroform-soluble contaminant. The dye content on the
label of a bottle of ethyl green should be close to 90%. NEVER use
a sample of "methyl green" that is not clearly identified by its
true CI number (42590) and preferably also its true name of ethyl
green. As to pyronine Y, the traditional Biological Stain Commission
requirement was a 45% dye content. Since the mid-1980s it has been
easy to obtain pyronine Y of higher purity and dye content. Only
more recently published techniques take this into account. The
instructions for a modern method will allow for the availability
of high quality ethyl green and pyronine Y.
For an EGP method that may meet the exacting standards of the
Eurocrats, see Hoyer et al (1986) Histochem J 18: 90-94. This is
perfectly straightforward, but it needs high-purity dyes. Older
batches, even if Certified, may not work.
The ethyl green-pyronine method is excellent for showing cells that
are rich in ribosomal RNA (red cytoplasm) in contrast to other
cells (bluish-green nuclei). In lymphoid tissue, plasma cells
stand out as occasional great red wheels among the greenish-blue
sea of lymphocyte nuclei. In the nervous system neurons have red
cytoplasmic Nissl substance and nucleoli, whereas glial cells
are seen generally as greenish nuclei - but with oil immersion
you can see red rings of RNA around the tight little green
nuclei of the oligodendrocytes. As with other Nissl stains, it
is difficult (perhaps impossible) to distinguish some small
neurons, as in the granular layer of the cerebellum for example,
from oligodendrocytes.
The only reason not to use the EGP method for everything is that
it does not impart any colour to collagen or to the cytoplasms
of cells that are not rich in rRNA.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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