Re: Cartilage staining
From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
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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