Safranin O for proteoglycan content in Articular Cartilage

From:Gayle Callis

Original publication is:

Rosenberg, L Chemical basis for the histological use of safranin O in the
study of articular cartilage.  J Bone and Joint Surgery, 53-A:69-83, 1971
This original publication used frozen sections of cartilage and NOT
paraffin embedded tissue. So how one interprets this stain for paraffin
must be used with care which leads to the next comments.  

Take care in choosing the correct fixative and decalcifying agent in order
to not extract proteoglycans.  EDTA extracts proteoglycans (biochemists
have been using this technic for years) and this changes the tinctorial
quality of the staining.  In other words, if proteoglycans are removed or
partially removed, the staining will be less intense and that can skew
results towards negative. It is good practice to have a piece of normal
undecalcified articular cartilage as a positive control to show any
extraction of proteoglycans by decalcifiers along with a piece of
decalcified cartilage. 

Kirviranta et al in Fixation, decalcification, and tissue processing
effects on articular cartilage proteoglycans Histochemistry 80:569-573,
1984 and others discuss this problem.  Eggert et al (1979 and 1981) found
that acidic buffers ie formic acid with either sodium formate or sodium
citrate were a better choices to prevent proteoglycan loss.        

A good review of articular cartilage staining is:

Getzy LL, Malemud CJ et al.  Factors influencing metachromatic staining in
paraffin embedded sections of rabbit and human articular cartilage: a
comparison of the safranin O and toluidine blue O technics.  J
Histotechnology 5:111-116, 1982.  Lee Getzy gave a wonderful workshop at
NSH convention some years ago on based on this work, complete with all
their methods.

Another excellent H&E stain for bone and cartilage is Ehrlichs hematoxylin
with eosin/phloxine. 

Sorry to blow the horn, but another publication: 

Callis G and D Sterchi in Decalcification of Bone: Literature review and
practical study of various decalcifyig agents, methods, and their effects
on bone histology. J Histotechnology (21(1):49-58, 1998 tested various
decalcifiers including EDTA, methods, etc and stained with Saf O and H&E to
show the differences in staining with the various decalcifiers plus an
extensive reference list.

Have a good weekend

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (voice mail)
406 994-4303 (FAX)


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