Re: Cartilage staining

From:"J. A. Kiernan" <>

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 experiment
  with times & concentrations: probably needs more time at lower
  concentration than for sections. Wash well with 1% acetic acid until
  no more colour comes out of the object, then soak in a dilute
  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 of
  the most stable of all organic compounds - unreactive and insoluble 
  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 resinous
  mountant (even if the latter did not dissolve or otherwise injure
  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, on
  sections rather than in bulk. Dilute eosin might do the trick
  in bulk. After counterstaining, keep in weakly acid solutions
  to avoid losss of dye, and make sure the aqueous mountant is
  also slightly acidified. (Slight acidification to prevent loss
  of an anionic dye means 0.01 to 0.1% acetic acid; no need to
  measure accurately).
  Just a few ideas! They are based on sound (I hope) principles
  that haven't, to my knowledge, been tested with your kind of
  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 to
  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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