Re: Counterstains for Vector VIP substrate

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From:Wendy Prime <>
Date:Tue, 27 Jul 1999 16:52:11 +0100 (British Summer Time)

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 
<> 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
$ > <> 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
$ > $
$ > 
$ > ----------------------
$ >
$ > Duncan Building
$ > The University of Liverpool
$ > Liverpool L69 3BX
$ > 
$ > Tel.0151 706 4503

Duncan Building 
The University of Liverpool
Liverpool L69 3BX

Tel.0151 706 4503

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