[Histonet] Re: damaged cartilage

[Robert Richmond]

About staining of damaged cartilage: A number of years ago I
researched and wrote up a procedure for a trichrome stain suitable for
staining normal and abnormal cartilage. I have never actually been
able to try it, but a researcher I gave it to said that it worked
satisfactorily.

Bob Richmond
Samurai Pathologist
Knoxville TN
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SAFRANIN O STAIN FOR CARTILAGE

PURPOSE: This highly specialized trichrome technique has been used as
a stain for normal and abnormal cartilage in osteoarthritis.

SPECIMEN: Tissue fixed in neutral buffered formalin, routinely
decalcified if necessary. Lillie used acidified formalin fixatives,
with perhaps better results.

PROCEDURE:

1.   Hydrate paraffin sections to water.
2.   Stain 6 minutes in WEIGERT'S HEMATOXYLIN.
3.   Wash well in water.
4.   Blue in dilute ammonia or other bluing reagent.
5.   Stain 3 minutes in FAST GREEN.
6.   Wash in 1% ACETIC ACID.
7.   Stain 12 to 15 minutes in SAFRANIN O.
8.   Drain sections, but do not wash in water.
9.   Dehydrate rapidly (about ten dips in each bath) through two
changes of 95% alcohol, two changes of absolute alcohol, and xylenes.
The alcohols should be fresh. Xylene substitutes should be evaluated
with caution.
10. Mount in resin (such as Permount).

Results: nuclei are black, cytoplasm gray-green. Proteoglycans in
normal cartilage stain uniformly red, while subchondral bone is
blue-green. Metachromatically staining substances such as mucins and
mast cell granules are orange-red.

REAGENTS:

WEIGERT'S HEMATOXYLIN: may be prepared or purchased. The shelf life of
the completely mixed iron hematoxylin solution is short, no more than
a few weeks. It might be possible to substitute an ordinary alum
hematoxylin here, if nuclear detail were of no great concern.

FAST GREEN:  Dissolve 40 mg of fast green FCF (C.I. No. 42053) in 200
mL of distilled or deionized water. Add a crystal of thymol or a drop
of phenol as a preservative. Expect a shelf life of a few months at
room temperature.

SAFRANIN:  Dissolve 200 mg of safranin O (C.I. No. 50240) in 200 mL of
distilled or deionized water. Add a crystal of thymol or a drop of
phenol as a preservative. Expect a shelf life of a few months at room
temperature.

ACETIC ACID: Add 1 mL of glacial acetic acid to 100 mL of distilled or
deionized water, or put about 8 drops in a 40 mL Coplin jar full of
water. Prepare fresh every day of use, unless the stain is done very
frequently.

QUALITY ASSURANCE: Use dyes obtained from documented sources. Examine
the slide for adequacy of staining. Use decalcified normal cartilage
and bone as a control.

SAFETY AND DISPOSAL:  These reagents offer no special safety or
disposal problems. They may be disposed of down the drain, with plenty
of water.

REFERENCES:

1. Lillie RD.  Histologic technic and practical histochemistry, 3rd.
ed., 1965, p. 507. Blakiston/McGraw-Hill, New York. The basic
procedure, developed by Lillie himself, is given here.
2. Rosenberg L.  Chemical basis for the histological use of safranin O
in the study of articular cartilage. J Bone Joint Surg 53-A (1):69-82,
1971. Another paper by Mankin et al. in the same journal, p. 523,
gives a histologic grading scale.
3. Hamerman D.  The biology of osteoarthritis. N Engl J Med
320:1322-30, May 18th, 1989. Color plates illustrate the desired
results of the stain.
4. Hamerman D., Dept. of Medicine, Montefiore Medical Center, Bronx
NY, Oct. 5, 1989. More details of the method.
5. Conn HJ, Lillie RD. Biological Stains, 9th ed, pp 385-7. Waverly
Press, Baltimore 1976. About safranin.
6. Prophet EB et al., eds. AFIP Laboratory Methods in Histology, p.
167. American Registry of Pathology, Washington DC 1992. Very short
procedure.

AUTHOR:
Procedure written by Robert S. Richmond, M.D., F.C.A.P., Knoxville TN,
October 1989. Bibliography updated June 1994.