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

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From:Margaret Gondo <>
To:"J. A. Kiernan" <>
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John -

I have attended a few seminars where someone will throw up a picture of
an 'injection site' and then say something about how they found the site
by nuclephil infiltration.  .  I know from experience that there are a
few techniques that can be localized beyond a shadow of a doubt just by
watching the change/damage in tissue (ie electromyography needles often
produce a focal neuropathy).  However, I'm always suspicious when
someone with little to no histology/pathology training says they can
localize an injection site based on cell infiltrate.I'm just curious as
to what your opinion is when someone says this.

Also, marking the general injection site area with a sharpie
marker/tattoo gun helps when the animal is sacrificed with the gross


J. A. Kiernan wrote:
> 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
>    E-mail:

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