Thank you for your collagen lesson.  Being a histologist in a orthopedic lab
for 18 years, I would never offer a partial procedure, so as you can see the
procedure I sent does advise the use of polarized observation, as well as the
published reference to help answer any questions that mightarise from the
procedure.  :~}

Karen Bowden
Research Associate II
University of CA, San Diego
9500 Gilman Dr.
La Jolla, CA  92093

"Tarpley, John" wrote:

> 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