Re: Fluoro-emerald-suitable counterstain

From:"J. A. Kiernan" <>

On Wed, 18 Oct 2000, Richard L. Young wrote:

> have marked the site (by iontophoresis) with fluro-emerald (D-1820, 
> Molecular Probes) and wish to visualise the site under confocal 
> and/or fluorescence micropscopy.

This product is a dextran conjugated with both lysine and fluorescein.
Adequate fixation with formaldehyde binds it permananently to the tissue.
> I am seeking advise for a suitable fluorescent or non-fluorescent 
> counterstain that will not quench any fluoroscence from the primary 
> marker. Does anyone have any experience with propidium iodide, 
> toluidine blue-O or such, and suitable protocol details to effectively 
> counterstain these critical brain stem slices (50 micron).

Propidium iodide (or ethidium bromide) will give you a Nissl-like
fluorescent (orange-red) stain. Try 1.0 mg/ml in PBS for 30 min,
then a few rinses in buffer and mount in a neutral aqueous medium.

Alternatively stain for 2 min in a 1:100 dilution of 0.5% neutral
red (C.I.50040; Basic red 5). Rinse in water. Air-dry thoroughly
(overnight at room temp), place in xylene for a minute or so and
apply a non-fluorescent resinous mounting medium (DPX, Cytoseal
or Entellan is OK). The fluorescence is similar to that of ethidium
& propidium (orange-red, optimally excited by green) but it neutral
red seems to have wider spectra of absorption and emission, and can
be seen with non-optimal filter combinations. It's a traditional
fluorochrome (also a very good red Nissl stain if used conventionally
at 0.5%) that has been largely forgotten in recent decades and
replaced by much more expensive compounds that do the same job.
A blue thiazine dye like toluidine blue will quench fluorescence of
objects that it stains. Thus, nuclei and Nissl substance often stand
out blackly against a fluorescent background of neuropil, tracts
etc. This probably is not one to go for if the section contains
fluorescently labelled neuronal somata.

Hope this helps.

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

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