USURPED. Vector NovaRed fading; Tests & thoughts

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From:"J. A. Kiernan" <>
To:Gayle Callis <>
Date:Tue, 29 Jun 1999 10:04:26 -0400 (EDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII

  Nobody would need to ask about this if the identity of
  the NovaRed product were not kept secret. 

  Probably the red end-product is either an anionic or a
  cationic dye. It should be easy to find out which, and
  then the remedy may be simple. Try the following.

  If it's an anionic dye it will be extracted from sections by
  mild alkali, such as a dilute solution of sodium or lithium
  carbonate or saturated calcium hydroxide. In that case, to
  preserve the colour wash in slightly acidified water (such
  as 0.1 to 0.2% acetic acid), shake & drain well, then
  dehydrate in 3 changes of 100% alcohol - it shouldn't matter
  which alcohol - and then clear and cover.

  If it's a cationic dye it will be extracted from sections by
  a weak acid, such as 1% acetic. I think this is unlikely,
  because many of the common counterstains used after an
  immunohistochemical staining are more acidic than this. 
  The people who developed NovaRed would know that, and would
  not have been so silly as to produce a chromogen that oxidized
  to an acid-soluble product. Just in case they _were_ silly, the
  way to prevent loss of the product would be: (a) avoid an acid
  counterstain - don't use an alum-haematoxylin or a basic dye
  to colour cell nuclei, for example. (Interesting alternatives
  exist, but not in the mainstream of "routine" methods.) 
  (b) Wash in slightly alkaline water (such as saturated
  calcium hydroxide or 0.1% lithium carbonate), shake & drain well, 
  dehydrate in 3 changes of 100% alcohol - it shouldn't matter
  which alcohol - and then clear and cover.
  Dehydration in 3 X 100% alcohol is preferable to a graded series
  because many (? most) anionic and cationic dyes are more easily
  extracted by lower alcohols (70% etc, even 95%) than by 100%,
  and you don't want to lose _any_ of the USURPED (Undisclosed
  Secret Unidentified Red Peroxidase End Product). 

  The experiment suggested above is explained rather long windedly,
  but should take only about 10 minutes to carry out. I can't do
  it myself because I haven't got any NovaRed, and am disinclined
  to do any more business with a firm that doesn't tell buyers what
  they are buying. That's a pity, because most of their stuff is well
  explained and not secret. All the Vector kits I've used have 
  worked well and have been accompanied by adequate explanatory

  When you buy a kit you pay the firm to relieve you of
  time consuming weighings, measurings and adjustments to the
  potency of intrinsically variable reagents. A good kit costs a 
  lot, and you buy it because you can't afford to do the same 
  development work yourself. Secret reagents are another kettle
  of fish. No respectable journal would publish the results of
  research done with unknown chemicals, and no sane pathologist
  would be foolish enough to use such substances for clinical
  or forensic diagnostic purposes. Just imagine the fun & games
  of cross-examination in the coroner's court. I could continue,
  but won't.
                             John A. Kiernan, MB, ChB, PhD, DSc 
                             Department of Anatomy & Cell Biology,
                             The University of Western Ontario,
                             LONDON,  Canada  N6A 5C1

On Mon, 28 Jun 1999, Gayle Callis wisely wrote:
> This question also went to Vector and in the process, answered itself
> as I type.  This new DAB (red color) substitute is wonderful, and 
> worked beautifully with acetone fixed mouse CD markers CD4, 8 , Mac1,
> and B220 but after rinsing in tap water, counterstaining and
> subsequent mounting in aqueous mounting media (Shandon Immunomount)
> totally faded within hours.  Bummer of bummers!  I guess aqueous mounting
> media is not acceptable with this chromogen, you need to dehydrate and
> use a regular mounting media.  Also, directions say to rinse sections
> for 5 min in water.  Question here:  running tap water?  distilled water?
> is this the source of fading also?  I know you need to avoid recycled
> alcohols with this chromogen.  It also says mounting with non aqueous mounting
> media acceptable, no mention of aqueous, but did not say no on the latter. 
> One good thing, NovaRed is so sensitive that antibody dilutions used with
> AEC chromogen were too concentrated, and a mouse CD4 at 1:1000 was still
> too concentrated.  Impressed with the sensitivity, but not the fading.
> Has anyone else had this happen?  or tried it with aq mounting media?
> So many questions, so little time!  
> Gayle Callis, 

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