On Wed, 30 Dec 1998, Karen D. Larison wrote to
peter.siesjo@neurokir.lu.se, and to histonet@pathology.swmed.edu

> True Blue has been used extensively by neurochemists to label retrogradely
> filled HRP-containing neurons.  In these experiments, ammonium molybdate is
> used to stabilize the signal.
 [Previously, sodium nitroprusside (=nitroferricyanide) had been used
  and it frequently caused non-specific deposition of needle-shaped
  blue crystals in the sections.]
> One commonly cited reference for this technique
> is Olucha, J Neuroscience Methods 13, 131-138 (1985).

  This paper described an improvement to the histochemical method
  for HRP-containing neurons using tetramethylbenzidine (TMB), a
  chromogen introduced by Rosene, Mesulam and colleagues in the 1970s.
  This method gives a blue product, similar to that obtained with
  variants of the traditional benzidine method, but TMB was said not
  to be a carcinogen. (Benzidine is, and DAB might be.) The blue
  product is much more photogenic than DAB-brown, even in B & W.

  True blue and a related substance called fast blue are "pet names"
  of 2 very strongly fluorescent dyes of the cyanine group. Their
  chemical names are long, awkward and impossible to remember. They
  are taken up by axon terminals and retrogradely transported to the
  cell-bodies of the neurons, where they can be seen in thick frozen
  sections of formaldehyde-fixed specimens. Commonly the sections
  are air-dried and either examined dry, or taken into xylene and then
  coverslipped with a non-fluorescent resinous mounting medium. These
  basic (= cationic) fluorochromes bind to DNA and RNA, providing a
  Nissl-like picture of each labelled neuron. Part of a local micro-dose
  of true or fast blue enters the general circulation, and the sensitivity
  of the method is so great that the circulating tracer sometimes enters
  all axon terminals that are outside the blood-brain barrier (in smooth
  and skeletal muscle, and in autonomic ganglia). Consequently, labelled
  motor and preganglionic neuronal cell bodies may be labelled in any
  tracing experiment using true blue, as may neurons that project to
  parts of the CNS where there is no blood-brain barrier.

  Conspicuous examples are the supraoptic and paraventricular nuclei
  of the hypothalamus. The neurohypophysis, which receives the axons
  from these nuclei, is outside the blood-brain barrier.

  Extreme sensitivity somewhat limits the usefulness of true blue and
  fast blue as retrograde neuroanatomical tracers. If anyone wants
  references for any of the stuff in the previous paragraphs, please
  email me personally. I used TMB and fast blue in some research work
  in the early 1980s (before the discovery of molybdate as a better
  TMB stabilizer than nitroprusside), and I encountered all the
  above-mentioned difficulties with the wanderings of true and fast
  blue.  I may well have missed important recent publications
  about these methods, and will welcome further enlightenment.

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