Sirius red staining must be read using polarized light otherwise the stain
will be over read since structures other than collagen stain red with light
microscopy. Perhaps the best reference for this point is Junqueira LCU,
Bignolas G, & Brentani RR: Picrosirius staining plus polarization
microscopy, a specific method for collagen detection in tissue sections,
Histochemical Journal  11:447-455, 1979. I think the original paper was
Sweat F, Puchtler H, & Rosenthal S: Sirius red F3BA as a stain for
connective tissue,  Archives of Pathology  78:69-72, 1964. In some of the
earlier papers after Sweat it was proposed that the color of the
birefringence was determined by the collagen type. Later papers showed this
not to be the case, but rather the color varies from green to yellow through
orange to red depending upon the corsslinking of the collagen which reflects
the age and fiber bundle size of the collagen since collagen is usually
first deposited as fine fibrils which later become crosslinked into larger
fibers and bundles.

John E. Tarpley 5-1-A
Associate Scientist
Amgen Inc.
One Amgen Center Drive
Thousand Oaks, CA 91320 

-----Original Message-----
From: rueggp [mailto:rueggp@earthlink.net]
Sent: Monday, October 08, 2001 9:36 PM
To: Karen Bowden
Cc: r-meyer2@northwestern.edu; histonet@pathology.swmed.edu
Subject: Re: sirius red for collagen


this is the same sirrus red protocol i use, except i do not store in fridge
and it
lasts for more than 6 months.
patsy ruegg

Karen Bowden wrote:

> I used this on rat pineal tissue and thought is looked great.  The
Investigator
> was expecting a lot less collagen so he didn't like it.  The collagen
stain red
> and the other tissue stained yellow just like the "results" said it would.
It
> is also a very easy stain.
>
> Fix specimen in 10% Buffered Formalin, embed in paraffin, section at 5
microns.
>
> 1. Clearing agent                 -      5 min.
> 2. Clearing agent                 -      5 min.
> 3. Clearing agent                 -      5 min.
> 4. 100% Ethyl Alcohol          -      3 min.
> 5. 100% Ethyl Alcohol          -      3 min.
> 6. 95% Ethyl Alcohol            -      3 min.
> 7. 95% Ethyl Alcohol            -      3 min.
> 8. 80% Ethyl Alcohol            -     3 min.
> 9. Tap water wash               -     3 min.
> 5. 0.1% Sirius Red                -     30 min.
> 15. 80% Ethyl Alcohol          -     1 min.
> 16. 95% Ethyl Alcohol           -     1 min.
> 17. 95% Ethyl Alcohol          -     1 min.
> 18. 100% Ethyl Alcohol        -     10 min.
> 19. 100% Ethyl Alcohol        -     10 min.
> 20. Clearing agent               -     3 min.
> 21. Clearing agent               -     3 min.
> 22. Clearing agent               -     3 min.
> 23. Mount with mounting media
>
> Results:
>  light microscopy: collagenous fibers - red
>  other tissue elements - bright yellow
>
>  polarized microscopy:  collagen fibers - orange/red bands
>  against black background
>
> 0.1% Sirius Red Solution
> Sirius Red dye 0.1 gm.
> Picric Acid saturated  100 ml.
>
> Store in refrigerator.  Stable for 4-6 months.
>
> Reference:  Greenberg, Stephen, Ph.D., Laboratory Medicine, Vol. 17, No.
8,
> August, 1986.
>
> Karen Bowden
> University of CA, San Diego
>
> r-meyer2@northwestern.edu 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
> >
> > Bob Meyer, HTL
> > Northwestern University