Re: Picrosirius stain. Possible causes of failure.

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From:"J. A. Kiernan" <> (by way of histonet)
To:histonet <>
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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

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