Re: Counterstains for Vector VIP substrate
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From: | Wendy Prime <W.Prime@liverpool.ac.uk> |
To: | kmerriam@tktx.com |
Reply-To: | |
Date: | Tue, 27 Jul 1999 16:52:11 +0100 (British Summer Time) |
Content-Type: | TEXT/PLAIN; CHARSET=US-ASCII |
Hi Kimberley
Your question prompted a discussion at home , "the man
indoors" was a histologist in a past life. Methyl Green is
rarley pure and in the back of my mind (and his ) I think
methyl green converts to Methyl Violet over a period of
time. I can not give a reference to back up my belief,
needless to say when we made up methyl green pyronin stain
we would keep the stain on a layer( 20ml or so) of
chloroform and this would extract the methyl violet as the
bottle stood on the shelf.
When i came to use methyl green again as a counterstain to
NBT-BCIP I extracted the methyl green in the same way.
Vector gave a recipe for methyl green in a pH4.? buffer. I
am sure they would give the formulation. By taking a 100ml
separation funnel, ( tear-drop shaped glass container with
tap at one end and a stopper at the other)close the the tap
at the bottom add 50ml of the the methyl green in the
aqueous buffer solution and then add chloroform to the
separating funnel, replace the stopper making the whole
unit liquid tight and gently roll the separation funnel
around in your hand You will see the methyl violet being
taken up by the chloroform. When the chloroform is very
purple loosen the tap and dispose of the contaminated
chloroform appropriately. Repeat the process by adding
fresh chloroform until the the chloroform no longer takes
up the methyl violet.
In the past this was the sort of procedure we did when we
wanted a quiet half hour. Now it is something i would
only undertake in the fume hood wearing the appropriate
protection. You may feel that this is a lot of hassle as
the solution to the problem but you may feel the procedures
worth it
As an aside we did use unextracted methyl green as stain
for amyloid. The "contaminating" methyl violet stained the
amyloid while the the methyl green acted as a nuclear
stain. This worked best on frozen sections.
On Mon, 26 Jul 1999 10:58:50 -0400 Kimberly L Merriam
<kmerriam@tktx.com> wrote:
$ Wendy,
$
$ Thanks for the information. I will try this, I did not know that you
$ needed to purify the methyl green. I purchased it from Vector,
$ shouldn't it be pure already?
$
$ Do you have the specific procedure for this purification step?
$
$ Kim
$
$
$
$
$ Wendy Prime wrote:
$ >
$ > Hi Kimberly
$ >
$ > Did you extract the contaminating Methyl Violet from the
$ > Methyl Green This is achieved by mixing the methyl green
$ > in an aqueous solution with chloroform, in a separating
$ > funnel. The metyl violet is extracted into the chloroform.
$ > This needs to be done a few times. It is best done in a
$ > fume cupboard for obvious reasons. It may be possible to
$ > otain pure methyl green from your dye supplier
$ >
$ > In the past I used methyl green with a NBT-BCIP kit it gave
$ > good results. The NBT product was blue black as opposed to
$ > purple, but the nuclei were always stained green.
$ >
$ > Regards
$ >
$ > Wendy
$ >
$ > On Fri, 23 Jul 1999 09:30:22 -0400 Kimberly L Merriam
$ > <kmerriam@tktx.com> wrote:
$ >
$ > $ Any suggestions for counterstain IHC sections stained with Vector VIP
$ > $ (purple) substrate? I have tried the recommended methyl green, but it
$ > $ seems to turn everything on the slide various shades of blue (including
$ > $ the cells that were stained purple prior to counterstaining).
$ > $
$ > $ Kim Merriam, MA, HT
$ > $ TKT
$ > $ Cambridge, MA
$ > $
$ >
$ > ----------------------
$ > wprime@liverpool.ac.uk
$ > CTBRC
$ > Duncan Building
$ > The University of Liverpool
$ > Liverpool L69 3BX
$ >
$ > Tel.0151 706 4503
----------------------
wprime@liverpool.ac.uk
CTBRC
Duncan Building
The University of Liverpool
Liverpool L69 3BX
Tel.0151 706 4503
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