Re: Methyl green
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From: | Robert Schoonhoven <rschoonh@sph.unc.edu> |
To: | Lynn Gardner <lynn-gardner@uiowa.edu> |
Reply-To: | |
Content-Type: | text/plain; charset=us-ascii |
Lynn,
My procedure is as follows, I have sent this one out a time or two and
no one has complained about it. It works very nicely in my lab..
Methyl Green Counterstain
REAGENTS:
a) Acetic Acid, Glacial (aldehyde-free)
b) Sodium Acetate, Anhydrous
c) Methyl Green
MIX
1 ) 0.1 N Acetic Acid: Add 6 ml of glacial acetic acid to 1000 ml of
filtered distilled water.
2) 0.1 N sodium Acetate: Add 4.102 g of sodium acetate to 500 ml of
distilled water.
3) Buffer:
a) Add 755 ml of 0.1 N acetic acid to 265 ml of 0.1 N sodium acetate.
b) Adjust pH to 4.2 with NAOH.
4) Methyl Green
a) Add 20.0 g of methyl green to the buffer solution.
b) Filter the methyl green solution.
STORAGE: room temperature
SHELF LIFE: Indefinite while in storage; when in use, the shelf life is
2 weeks or until staining becomes too weak.
PROCEDURE:
1) After color development of slides, rinse in distilled water briefly.
2) Place slides in methyl green solution for 3 to 5 minutes, or as long
as necessary to get the
staining desired. (slides which have been denatured, i.e. as
for in-situ-hybridization,
typically need far less Counterstain 30 seconds or so.)
3) Do not rinse in water. The methyl green washes out too easily. Go
directly into 95%
alcohol for a brief time (about 10 dips) .
4) Continue dehydrating through one more 9S% alcohol, then 3 changes of
100% alcohol,
allowing the slides to stay in the last alcohol for 2-3
minutes.
5) Clear through 3-4 changes of xylene, and coverslip.
Lynn Gardner wrote:
>
> Hey all, I know people have been discussing this lately however, I didn't
> pay close enough attention.
>
> I am in need of a methyl green counterstain for alkaline phosphotase
> stained tissue that will be crisp and consistant and won't decrease in
> intensity over time. Does anyone know of such a critter???
>
> Thanks all
> Lynn Gardner
--
best regards,
Bob
Robert Schoonhoven
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina
CB#7400
Chapel Hill, NC 27599
Phone
office 919-966-6343
Lab 919-966-6140
Fax 919-966-6123
Don't go around saying the world owes you a living; the world owes you
nothing; it was here first.
Mark Twain [Samuel Langhornne Clemens] (1835-1910)
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