RE: Cartilage staining

I use Azure B for cartilage staining, 0.2% solution in Dih20, ph 4.
This can be reused for months.  Stain sections for 5 min. rinse in water and
air dry, coverslip dry with permount, don't go through alcohols and xylene
the stain will be removed.
Patsy Ruegg

		-----Original Message-----
		From:	J. A. Kiernan []
		Sent:	Monday, November 13, 2000 10:48 PM
		To:	Tahir Mahmood
		Subject:	Re: Cartilage staining

		On Mon, 13 Nov 2000, Tahir Mahmood wrote:

		> I have a question about problems i am having with staining
cartilage. The
		> biggest issue is that the tissue has been grown on
synthetic polymers, and
		> the polymer itself swells considerably. It is also soluble
in xylene. So
		> "regular" safranin O staining results in 1. the overall
matrix to implode,
		> and 2. whatever polymer was once there is dissolved (I
need to see the
		> polymer).

		  How about staining the formaldehyde-fixed preparation
whole with a
		  dilute solution of alcian blue at pH 1?  You might need to
		  with times & concentrations: probably needs more time at
		  concentration than for sections. Wash well with 1% acetic
acid until
		  no more colour comes out of the object, then soak in a
		  ammonia or other alkaline solution (this converts the
bound alcian
		  blue to copper phthalocyanine pigment (CuPC). CuPC, which
also has
		  several non-chemical names, such as monastral blue, is one
		  the most stable of all organic compounds - unreactive and
		  in pretty well everything. It is, in fact, the final
product of any
		  staining (or dyeing of fabric) with alcian blue. 

		  Your specimen could then be sectioned (cryostat, freezing
or vibrating
		  microtome, if solvents attack the substrate) and mounted
onto slides
		  using any aqueous mounting medium. There will be no
trouble with
		  bleeding of the dye into the medium, which cab be
troublesome with
		  all basic dyes (includung safranines) other than alcian
blue. The
		  rather low refractive index of nearly all aqueous
mountants may 
		  allow greater visibility of your polymer substrate than a
		  mountant (even if the latter did not dissolve or otherwise
		  the stuff). 

		  If your polymer substrate is a polycation, you might be
able to
		  counterstain it with a red or yellow anionic dye or both. 
		  van Gieson or picro-sirius red would be good ones to try,
		  sections rather than in bulk. Dilute eosin might do the
		  in bulk. After counterstaining, keep in weakly acid
		  to avoid losss of dye, and make sure the aqueous mountant
		  also slightly acidified. (Slight acidification to prevent
		  of an anionic dye means 0.01 to 0.1% acetic acid; no need
		  measure accurately).
		  Just a few ideas! They are based on sound (I hope)
		  that haven't, to my knowledge, been tested with your kind
		  cultures. All these suggestions are based on well studied
		  published methods and my own experiences applying these to

		  sections and a variety of whole objects, including primary
		  explant organ cultures. If you would like some references
		  books, papers etc, just ask. 

		 John A. Kiernan,
		 Department of Anatomy & Cell Biology,
		 The University of Western Ontario,
		 LONDON,  Canada  N6A 5C1

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