Histology FAQ

Staining, Histochemistry and Histotechnology

(Frequently Asked Questions)


Dr. John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London, Canada



FAQ Home > Staining Methods, Histochemistry


Evans blue, trypan blue and eosin as tracers


Can Evans blue be used as a tissue dye, and will it safely wash out of the tissue during routine paraffin processing?  The object is to trace a catheter leakage then have the dye wash out of the tissue during processing. Would eosin be OK for the same purpose?

Answer 1.

Evans blue is an anionic dye with large molecules, closely related to trypan blue. It was formerly used (? still is in some places) to measure blood volume, because it binds to serum proteins and stays in the circulation for a few hours. When it leaves the blood, some of it sticks to collagen (the elongated dye molecule favours this) and some is taken into cells, including macrophages and neurons. The dye-protein complex is fluorescent (red emission) and this was the first fluorescent tracer of neuronal uptake and retrograde axonal transport.

Applied to sections, trypan blue stains everything and can be washed out completely. Slight alkalinity speeds up the procedure. In the presence of another anionic dye with smaller molecules (like picric acid), trypan blue becomes selective for collagen, but is no match for acid fuchsine or sirius red F3B. I'm sure Evans blue, which is a VERY similar compound, would have identical properties as a stain.

So:  if you want to get rid of the Evans blue, wash the specimens in slightly alkaline water.

Eosin could also be used in the same way. If you're after very small leaks from your catheters, eosin might be more sensitive, because it's quite strongly fluorescent even without binding to anything (green-yellow emission). You could turn off the lab lights and use a Woods light to watch for leaks. Eosin is also removable by slightly alkaline water or by alcohols. Acidic reagents precipitate the insoluble colour-acid.

John Kiernan
London, Canada

Answer 2.

Evans blue and trypan blue both can be used to determine cell vitality - live cells exclude the dye(s), dead cells take then up - the trypan (Evans) blue exclusion test.

As far as catheter leakage is concerned, a fluorescent dye would certainly be a good choice.  Cavers use them to trace underground rivers, and fluorescent dyes are used for a similar purpose in opthalmology.

Russ Allison, Wales