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