Re: fast red (neutral or nuclear?)

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From:"J. A. Kiernan" <jkiernan@julian.uwo.ca>
To:"Molinari, Betsy" <BMolinari@heart.thi.tmc.edu>
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> Is neutral red the same as nuclear fast red? I am doing a Perls Iron
> stain and have two protocols one says  nuetral red and the other nuclear
> fast red as counterstain. 

  No.  Either is a good counterstain for Perls' method, but the
  counterstained things will not be the same.

  A short account of these two dyes follows. I don't give
  references for what I say, because it's nearly all old stuff and
  can be found in standard reference works and even in textbooks.
  My opinions (as distinct from facts) are, I hope, clearly
  separated from the purely informative statements.   

  Neutral red is a simple cationic (basic) dye. Used at pH 3 to 4
  it will stain nuclear chromatin (due to DNA and nucleolar RNA),
  cytoplasmic RNA (Nissl bodies, ergastoplasm etc, corresponding to
  ribosome-rich parts of cytoplasm) and various carbohydrate
  secretory products and extracellular materials. The sulphated
  carbohydrates, including mast cell granules and cartilage matrix,
  are commonly metachromatically coloured orange. The orthochromatic
  colour with neutral red is a bold scarlet.

  Neutral red (C.I. 50040) is used for several purposes and has been
  available as a "Certified" dye for many (60 or 70, I think) years.
  Its solutions keep well (shelf life at least 10 years in my
  experience), and you can water it down if the level in the bottle
  or staining tank is low, with little or no consequent prolongation
  of the staining time. This is one of the stains that I rate in
  the 5-stars (out of 5) category, because it always works, never
  lets you down even when you misuse it, keeps for ever, has a
  pleasing appearance, and provides useful information about tissue
  structure even when used alone. On top of that, it can be used
  in greatly diluted solution to make a fluorescent cationic stain
  or counterstain for nuclei and other basophilic things. Optimum
  excitation is by green light (orange-red emission with a 
  rhodamine-type filter set-up), but you can also see it with 
  broad-band UV or blue excitation. The use of neutral red as a
  fluorochrome is not recent (50+ years old), but the dye is better
  known for its applications in ordinary light microscopy.   

  Nuclear fast red, also sold under its German name of Kernechtrot
  (which means the same thing) is an anionic dye that is used in
  conjunction with an aluminium salt as a mordant. It works in
  much the same way as an alum-haematoxylin, ideally giving selective
  staining of nuclear chromatin, which is not (or only partly) due to
  nucleic acids. In practice there is often a pale pink nonspecific
  background coloration, which may be an annoyance or somewhat
  helpful. The colour has a hint of blue in it, and might be called
  crimson rather than scarlet. (It resembles staining with carmine
  or one of the alum-carminic acid methods.)

  There are some bottles labelled nuclear fast red that probably
  contain something quite different. If you buy new, it should be
  OK if the C.I. number 60760 is included in the information in
  the catalogue and on the bottle label. If we're giving stars to
  stains, I'd award this one 2, along with Mayer's carmalum. This
  compares with 3 or 4 for alum-haematoxylins. 
 
  A better purely nuclear stain that is red is alum-brazilin. You 
  simply substitute brazilin for haematoxylin in your favourite 
  brew, and come up with Mayer's brazalum etc. Use it in the same 
  way as a haemalum, but the nuclei are red instead of blue. 
  The brazalum red is close to the neutral red colour, which I 
  call scarlet, possibly incorrectly. It's the colur of British and 
  Canadian mailboxes: pillar-box red. When a red colour is shifted 
  towards purple, I use the word crimson, following the example of 
  botanists when they state the colours of fruits. 
  
  Brazilin is greatly neglected and has become a rare and expensive
  chemical. I could say more but won't, because this email is
  already too long. Perhaps another time. There should be some
  information about brazilin in the HistoNet archives from about
  three years ago.

  Bottom line(s).

  Counterstain Perls with neutral red if you need the red stain
  to locate the Prussian blue deposits accurately. Use nuclear
  fast red if you must show iron, nuclei and nothing else.


 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1






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