Re: Evans Blue (& other fluorescent counterstains)

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From:"J. A. Kiernan" <> (by way of histonet)
To:histonet <>
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On Mon, 19 Oct 1998, Barry, Lilith wrote:

> In previous email correspondence it was suggested by few people to use Evans
> Blue stain as a counterstain on FITC labelled sections. Is there a special
> protocol for it.

  The advice to use Evans blue probably came from a research worker
  who had experimented intelligently and come up with something useful
  for his/her purposes. If you want to use this or any other dye as a
  counterstain for immunofluorescence, you will have to try and err.
  There is no such thing as a "protocol" for this sort of thing.

  Evans blue and similar dyes (notably trypan blue) are not fluorescent
  but they become fluorescent when they bind to some (but not all)
  proteins. If you stain a section with 1% Evans or trypan blue at
  any reasonable pH (meaning 3 to 7), you get everything in a weary
  blue colour, with some feeble fluorescence here & there. The
  fluorescence can be seen (weakly) with rhodamine-type setups
  (green excitation; orange emission) or with broad-band UV and a
  colourless barrier filter (looks yellow). If there's too much
  blue dye that's not protein-bound, it quenches all the fluorescence,
  as do most blue dyes.

  If you need a counterstain to show you where you are in an
  immunostained section, I'd recommend a weakly fluorescent basic
  dye, which will show nuclei, cytoplasm of RNA-rich cells, and
  acidic carbohydrates (cartilage matrix, some mucus, etc).
  Neutral red does this pretty well: 0.001 to 0.01%, at or
  near pH 4. By changing  the filter block you can vary the
  relative prominences of this dye and various other
  fluorochromes. Try various concentrations of
  neutral red on any old sections to decide on an ideal
  time and concentration for this fluorochrome.

  It is not possible for anyone to provide a "protocol" for
  fluorescent counterstaining. The technique varies with
  the requirements of the investigation.

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

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