Re: Sirrus vs sirius (Van Gieson variants)

From:"J. A. Kiernan" <>

On Fri, 18 May 2001, Connie McManus wrote:

> What procedure are you using?  This sounds like van Gieson's, but all my
> procedures say to use 1% acid fuchsin or ponceau S.  Conn's says, however,
> that sirius red can be used in place of acid fuchsin for the van Gieson's
> stain and that "it resists fading better than the acid fuchsin and it gives
> a strong, selective staining of reticulum, basement membranes and collagen
> when used to replaced with acid fuchsin." (See p. 199 Conn's Biological
> Stains, 9th ed).

Right on the ball! The late R.D.Lillie (1896-1979) made important
contributions to connective tissue staining and developed a number 
of methods that used dyes other than acid fuchsine to stain collagen 
from solutions in saturated aqueous picric acid. Lillie's 
picro-aniline blue methods are just as easy to use as Van Gieson
and they stain thinner collagen fibres and basement membranes. 
Picro-sirius red does this too, and has the added advantage of
greatly increasing the birefringence of collagen fibres (see refs
in an email to histonet yesterday).

Lillie wasn't the first, either. Cajal's trichrome dates from ?1900. 
(It isn't a trichrome in the modern sense of the word, because it 
doesn't include phosphomolybdic or phosphotungstic acid.) It consists
of a strong nuclear stain with basic fuchsine followed by
indigocarmine (a blue dye) dissolved in good old sat. aq. picric
acid. I did a lot of these in the early '80s on rat intestine
that had been transected and surgically repaired. The results
were spectacular: tetra- rather than trichrome, with really
strong red nuclei, blue for dense collagen (submucosa), yellow
for erythrocytes, and green for cytoplasm of smooth muscle and
other cells. A mercuric chloride containing fixative was needed
for this colour scheme. After formaldehyde the blues and yellows
merged into green, and there was a general loss of brightness

Lillie's picro-aniline blue is very tolerant of simple formaldehyde
fixation, and I don't know why it isn't more widely used. Although
less beautiful than Cajal's trichrome, the resolution of collagen
fibres is better. Lillie's "allochrome" method, which is iron 
haematoxylin for nuclei followed by PAS and picro-aniline blue, 
is another cleverly contrived method that doesn't seem to be used 
much, despite being very good.

With picro-aniline blue, picro-indigocarmine or picro-acid fuchsine
you don't get enhancement of collagen fibre birefringence (in fact,
it's reduced somewhat). For this effect you need a dye with long
molecules, and sirius red F3B holds the record among the dyes used
in histology (it's a tetra-azo dye). Trypan blue is quite long,
and causes some enhancement. Puchtler studied many different dyes
when she developed this technique, and sirius red was the best.
It's also the slowest (needs 30-60 min whereas aniline blue etc need 
only a minute or two).

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

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