Re: live tissue stain (black, cyan, fluorescent)

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From:"J. A. Kiernan" <>
To:Judy Trogadis <>
Content-Type:TEXT/PLAIN; charset=US-ASCII

On Wed, 2 Aug 2000, Judy Trogadis wrote:

> We are trying to inject a compound into a live mouse.
> After 2 days the animal will be sacrificed and histology
> performed.  To ensure that the injections reached the
> appropriate site, we want to include a dye with the fluid
> for identification.
> Does anyone know of a stain which would be easily
> visible yet not interfere with the normal metabolism
> of the cells.

  You need a label (= tattoo) that gets phagocytosed at the
  injection site by cells (macrophages) that do the job
  and then do no more wandering.

  The traditional answer is India ink (make sure you have
  the real thing), which is extremely tiny carbon particles.
  They end up in macrophages, so the injection site is
  permanently labelled (tattooed) by black cells. A small
  bottle of genuine India ink (the trade name Pelikan comes
  to mind; I'm sure there are others) may cost $10 or so
  these days, but it will last for ever. If you use 
  2 microlitres 5 times a day a 50 ml bottle will still
  be more than half full after 7 years (allowing for an
  uncalculated 5% loss of volume by evaporation, and assuming
  that you unscrew the cap 2000 times without knocking the
  bottle over).

  Black may not be a good colour for your injection site label.
  This will be the case if you are injecting into fat and
  intend to stain with sudan black B, or for almost any tissue
  that will be stained with osmium tetroxide or a silver, gold
  or palladium reduction method, or with iron-haematoxylin.
  For these you need a non-black label. A currently popular
  one is monastral fast blue (C.I. Pigment blue 15). This is
  pigment-grade (= finely particulate) copper phthalocyanine,
  which is an extremely stable and insoluble substance that
  moves around and gets phagocytosed in much the same way as
  the colloidal carbon in India ink. It's colour is turquoise.
  I can send you some references if you're interested. This
  pigment is a major article of commerce (paints, inks, plastic,
  etc etc) and it isn't very expensive when you buy it as a
  laboratory chemical. As for India ink, make sure you buy a
  finely particulate pigment; not chunks or big crystals.

  There are also fluorescent plastic microspheres (available
  in various sizes and with various chemicals on their surfaces
  that make them edible to various cell-types, including
  macrophages). These are sold as suspensions, in small volumes
  (1 ml would be a big volume). They will show the injection
  site as bright cells on a dark background. This will be
  valuable if you can show fluorescence that's significant to
  your work in a contrasting colour. 

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

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