RE: Cartilage staining
From: | Patsy.Ruegg@UCHSC.edu |
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 [mailto:jkiernan@julian.uwo.ca]
Sent: Monday, November 13, 2000 10:48 PM
To: Tahir Mahmood
Cc: histonet@pathology.swmed.edu
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
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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