Re: Picrosirius stain. Possible causes of failure.
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> (by way of histonet) |
To: | histonet <histonet@magicnet.net> |
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On Tue, 20 Oct 1998, Martha Strachan wrote:
> Was wondering if anyone has some insight into my staining problem with
> Picrosirius Red. I'm using a protocol from Junqueira et al., J Path,
> 1986. The specimens are decalcified paraffin embedded bones. Most of
> the tissue elements are showing up pink with diffuse red throughout.
> I'm pretty sure the picric acid is saturated (21g to 1L) and the
> solution is cloudy. The protocol calls for 1 hour in 0.1% Picrosirius
> followed by a tap water wash
This could be extracting bound dye. For this method and
van Gieson, I'd recommend washing in 0.1% acetic acid.
This removes only the unbound anionic dyes.
> ... until no color is visible in the wash and
> staining with harris hematoxylin after the wash.
Not advisable for the same reason. The blueing of an
alum-haematoxylin needs neutral or slightly alkaline
water, and this will extract anionic dyes.
The traditional (and best) nuclear stain is Weigert's
iron haematoxylin, done before the van Gieson or
picro-sirius red. If you must have a blue nuclear
stain, iron-chromoxane cyanine R is OK. Haemalums
don't stand up well to acid counterstains like the
picro- mixtures (pH 1.3-1.6).
With polarizing microscopy the nuclear stain is often
omitted, because you don't see the nuclei even if they
have been stained.
> .. I was under the
> impression that the stain should look similar to a van Gieson but with
> better polarizing properties.
That's right. It can pick out collagen fibres and basement
membranes that are thinner than can be stained with van G, and
some people say picro-sirius red better keeping properties too.
(Basement membranes, unlike fine collagen fibres, don't become
birefringent - wrong type of collagen.)
Another thing that could be wrong is the red dye. It must
be sirius red F3B (CI 35780; Direct red 80). There are other
sirius reds around that are quite different and won't work
for this method.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
Phone: (519) 679-2111
FAX (Department): (519) 661-3936
E-mail: kiernan@uwo.ca
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