[Histonet] Recent issues with picro sirius red staining (entire liver section become red, no yellow background)

[abi jag]

Hello Histonetters,I am writing this to seek your help regarding a very recent problem that I am currently facing with Picro Sirius red staining of lab animal (mouse and rat) liver samples. I follow the procedure that was provided by John Kiernan in the histonet archives (please see below), which was working very well. Quite recently, the complete section become stained as red. Usually, collagen in the sections get stained as red with a yellow back ground. Please note that there was no change in the procedure/reagents etc, It will be of great help if you help me in troubleshooting this issue.With my best regards,Abijag
Sirius red collagen procedure

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Sirius red collagen procedure


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Solution A. Picro-sirius red

  Sirius red F3B (C.I. 35782):     0.5 g
  Saturated aqueous solution
    of picric acid:                500 ml
  Add a little solid picric acid to ensure saturation
    (This is important).

  (Keeps for at least 3 years and can be used many times.)

Solution B. Acidified water

  Add 5 ml acetic acid (glacial) to 1 litre of
  water (tap or distilled).

Procedure

Fixation is not critical, The method is most frequently used on
paraffin sections of objects fixed adequately (at least 24 hours
but ideally 1 or 2 weeks) in a neutral buffered formaldehyde
solution.

1. De-wax and hydrate paraffin sections.
2. (Optional, and not usually done) Stain nuclei with
   Weigert's haematoxylin (as for the van Gieson method,
   but more strongly, then wash the slides for 10 minutes
   in running tap water).
3. Stain in picro-sirius red (Solution A) for one hour.
   (This gives near-equilibrium staining, which does not
   increase with longer times. Shorter times should not
   be used, even if the colours look OK.)
4. Wash in two changes of acidified water (Solution B).
5. Physically remove most of the water from the slides
   by vigorous shaking or (for a few slides only)
   blotting with damp filter paper.
5. Dehydrate in three changes of 100% ethanol.
6. Clear in xylene and mount in a resinous medium.