Re: [Histonet] Re: Proteoglycan - decalcification - Why safranine?

From:Kim Merriam

At my last job, we were looking for retention of glycosaminoglycans, in both soft tissue and bone.  We would fix the tissue in methacarn (methyl carnoys) and decal in 10% formic acid containing ion exchange resin.  EDTA will cause a severe decrease GAG retention in the bone (we tested this decal method for GAG retention, and it was amazing how different it was than with the formic acid decal method).
 
Good luck,
 
Kim Merriam 
Novartis
Cambridge, MA

John Kiernan  wrote:
Dear Dr Balaji,

Adequate fixation is important before you decalcify
anything. That means at least 24 hours (a week is probably
better) in a neutral buffered (or any other) formaldehyde
solution.

Is your "Saf/FG" method safranine O followed by fast green FCF?

There are many such methods. One group is, to plant anatomists,
what H&E is to animal pathologists. (I apologize for using nouns
as adjectives, implying that anatomists are plants whereas
pathologists are animals.) The other lot of safranine-fast green
methods are the stuff of traditional light microscope cytology,
for showing chromosomes and cytoplasmic organelles in truly thin
paraffin sections of tiny bits of tissues fixed in solutions
containing osmium tetroxide and potassium dichromate. 

I have used both groups of methods. The plant anatomy ones are
straightforward if you follow the instructions, which include
checking with a microscope before making the preparation a
permanent one - just like H&E(!). 
(My limited trials of safranine and fast green FCF for 
traditional cytology were all dismal failures; the colours 
initially seen were always largely lost in the dehydrating 
alcohol.) 

If you need to stain proteoglycans in cartilage, why not use
an easy staining method that does not involve critical
differentiations? There are many such methods. In cartilage,
they are very clean and selective. Sulphate ions
(in the chondroitin sulphate glycosaminoglycans of the
proteoglycans) bind cationic dyes at pH 1.0 or even lower.
Alcian blue at pH 1 can be followed by any counterstain
because alcian blue changes into a permanently insoluble
pigment while the unbound dye is being washed out of the
sections.

Carbohydrate histochemistry is a large subject. It
includes many inexpensive but discriminating techniques
that are neglected by well funded researchers and clinical
pathologists. 

John A. Kiernan
London, Canada.
_______________________________________
balajimr@drreddys.com wrote:
> Thank you very much for your replies to my earlier mails. This is with
> regard to Saf/FG staining of cartilage. I want to demonstrate
> proteoglycans in cartilage of femur joint, tarsal joint and ankle joint. I
> am in the process of standardization of the same. I have taken a trial
> tissue of joints from Rat and I have used 20% EDTA for decalcification. I
> read somewhere that it affects the proteoglycan staining. Is it true? If
> so could you please suggest a good decalcifier for the same.Also let me
> know how long I can fix these joints for good fixation in NBF. Thanks in
> advance.
> 
> Dr. Balaji
> Dept. Pre clinical safety evaluation,
> Discovery research,
> Dr. Reddys Laboratories ltd. Bollaram Road,
> Miyapur, Hyderabad, 500 049
> Andhra Pradseh, INDIA
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