Re: sirius red for collagen (rather long)

From:"J. A. Kiernan"

On Mon, 8 Oct 2001 wrote:
> Has anyone done a sirius red stain for collagen?  If so, could I please have 
> your protocol.  The AFIP histology manual shows a sirius red method for 
> amyloid.  But the researcher that gave me this project says he is not looking 
> for amyloid but collagen.  I mentioned trichrome but I guess he has already 
> tried this.  Thanks 

Dear r-meyer2,

The method your researcher is after is picro-sirius red.
This mixture is used instead of van Gieson's stain (which
is in all the books). The only technical difference is
that you must stain for 30 minutes (30 seconds is often
enough with van Gieson). I recommend a post-staining
rinse in weak (e.g. 0.1-0.5%) acetic acid, then dehydrate
in 3 X 100% alcohol, not a graded series. This maximizes
retention of sirius red by the thinnest collagen (reticulin)
fibres and basement membranes.

The staining solution is 0.1% sirius red F3B (CI 35780; 
Direct red 80) in saturated aqueous picric acid. It keeps
for at least 4 years and you can use it repeatedly. Be
sure to get the correct dye. Add a bit of picric acid
sludge to the bottle to ensure saturation. Filtering
is not needed, in my experience.

The researcher must know the advantages and limitations
of the technique vis a vis the original van Gieson or the
various trichrome methods, or he wouldn't have asked you
to do this particular technique. Two good references are:
Puchtler et al 1973 Beitr. Pathol. 150:174-187 and
Junqueira et al 1979 Histochem. J. 11:447-455. Read at
least one of these to get a proper understanding of this
excellent technique and get the best out of the results. 
For some research purposes it is desirable to use a lower
concentration of sirius red (see Canham et al 1999 Neurol
Res. 21:618-626). 

About 4 years ago someone (I've forgotten who) posted 
to Histonet an excellent commented bibliography of 
picro-sirius red from which I, for one, learned a lot. 
It should still be there in the Archives 

Oh! for the heady days when Histonet was young, and 
every unscriber's attempt to unsuscribe or unsubsribe 
was revealed to all. Was it on a 1st of April
that Russ Allison wrote an uncompiled statistical
survey of the unsriptory variants?


In some research applications the initial staining of 
nuclei with Weigert's iron-haematoxylin is not needed 
and is omitted. If you include the nuclear stain you 
should overdo it because 30 mins in the quite strongly 
acidic picro-sirius mixture causes significant
extraction of iron-haematoxylin. If you initially get 
a nice selective nuclear stain, as when doing van Gieson's
method, it will be unpleasantly weak (though still
selective) after 30 minutes in picro-sirius red. As with 
nearly all other staining methods, understanding and 
judgement are needed to get the best results for the 
job at hand. 

In terms of "difficulty" I'd rate picro-sirius red alone 
as extremely easy (after one or two trial runs), at the
same level as uncritical PAS and alcian blue methods. 
Iron haematoxylin & picro-sirius red is less difficult 
than getting a good (as distinct from mediocre) result 
with haemalum & eosin (H&E). It's difficulty rating is 
perhaps the same as Mallory's or Masson's trichrome, and
easier than Heidenhain's AZAN trichrome. (Silver methods
for reticulin are in a higher class of difficulty than any
of the dye-based techniques.)

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

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