Two details are not clear from your method.

1. Does the picro-sirius red solution contain
   some undissolved picric acid (deposit in
   bottom of bottle)? It should, to ensure
   saturation.
2. Is there an intervening wash between dyeing
   in the picro-sirius solution and the first of
   the 3 changes of 100% alcohol? You should not
   wash in water, but slightly acidified water
   (eg approx 0.1% acetic acid) is desirable to
   rinse away only unbound dyes. Water that is
   not acidified will remove some sirius red, and
   you may lose some finer fibres, basement membranes
   etc.

As you have probably noticed, nuclear staining by
Weigert's iron-haematoxylin is weakened by subsequently
applied picro-sirius red (in contrast to Van Gieson's
picro-acid fuchsine). This is because of the longer
time in an acidic medium (30 min in contrast to less
than 5 with van G). For your purposes the nuclear stain
seems pointless. With your rapid dehydration you are
conserving the yellow cytoplasmic (muscle) stain, so you 
should be able to measure the total area and the
red (collagen) area. Picric acid is rather easily
removed by alcohol; most users of the picro-sirius red
method do not mind this, or even prefer an unstained
cytoplasmic background.

If you are using picro-sirius red for research 
purposes you should be familiar with the classic
papers of Puchtler and of Junqueira from the 1960s
and 1970s. A Google search for Puchtler and Junqueira
brings up some 25 items; several are Histonet
replies with technical instructions and lists of
references to get you started in the library.
-- 
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/
   http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________
bettina.hutz@orionpharma.com wrote:
> 
> ello,
> 
> we have some difficulties with PSR staining and so far no trouble = shooting
> advice helped us finding the reason for our problem. We use PSR as routine
> staining in CV reserch studies in order to = measure the quantitive amount
> of connective tissue in heart slides. Therefore the staining has to be
> proper with a big contrast between yellow and red = areas. In the beginning
> all went well, but now the staining quality has = decreased, the connective
> trissue is not as bright stained as for example one year = ago. I use the
> same batch of picric acid and Pirco Siriusred and have not = changed
> anything in the staining procedure.=20 Does anyone has any idea what can be
> the reason? My staining instruction here:
> 
> PSR (Picro-Sirius Red) staining of paraffin slides
> (modified by Bettina Hutz and Ritva Inkinen)
> 1.      Staining procedure
>         Deparaffination
>         3x xylene                               each min. 3 minutes
>         2x ethanol 100%                         short
>         ethanol 90%                             short
>         ethanol 80%                             short
>         ethanol 70%                             short
>         aqua dest.                              Short
>         Staining
>         Weigert=B4s Hematoxylin         10 minutes
>         Running tap water                       10 minutes
>         Rinse with aqua-dest.
>         Picro-Sirius Red                        30-60 minutes
>         Dehydration & mounting
>         3x ethanol 100%                 short
>         2x xylene                               short
>         Mount with xylene based medium
> 2.      Solutions
> 2.1.    Weigert=B4s Hematoxylin
> 
> 2.1.1   Stock solution A
> Hematoxylin (Natural Black 1, C.I. 75290)       10,0 g
> Ethanol 95%                                     1000 ml
> Produce app. 2 weeks before use
> 
> 2.1.2.  Stock solution B
> Ferric chloride, 29% aqueous            40 ml
> Aqua-dest.                                      950 ml
> HCl conc.                                       10 ml
> 
> 2.1.3   Usage solution
> Equal parts of solution A and solution B, use immediately.
> 2.2  Picro-Sirius Red
> 
> 2.2.1   Stock solution
> Sirius Red F3BA                                 5,0 g
> Picric acid, saturated, aqueous         500 ml
> Allow to stand for 48 hours before producing the usage solution.
> 
> Thanks for your help:)
> 
> Bettina Hutz
> 
> Bettina Hutz
> Research Assistant
> Department of Discovery Biology
> Orion Corporation, ORION PHARMA
> Orionintie 1, P.O.Box 65, FIN-02101 Espoo, Finland
> Tel. +358 10 429 2607,  Fax +358 10 429 2924
> Email: bettina.hutz@orionpharma.com
> 
> _______________________________________________
> Histonet mailing list
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