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

[John Kiernan]

I've never seen the kind of staining you describe, abi jag, with "the complete section become stained as red" but I've never used the method on sections of liver.

You should get red collagen and yellow hepatocytes with blue nuclei. The strongly acidic picrosirius stain, applied for an hour, always greatly weakens (differentiates) a prior nuclear stain with Weigert's or Lillie's iron-haematoxylin.  It's also quite easy to lose some or all the yellow in the washing and dehydration of the stained sections. Most users of this method are interested only in collagen fibres and do not mind if the nuclear and cytoplasmic colours get lost. The iron-haematoxylin nuclear stain is often omitted.


It is necessary to have the right dye. It must be sirius red F3B (= CI 35780 = Direct red 80). This is still used as a textile dye, with several suppliers and trade-names. See http://www.worlddyevariety.com/direct-dyes/direct-red-80.html#respond

Direct Red 80 - worlddyevariety.com<http://www.worlddyevariety.com/direct-dyes/direct-red-80.html#respond>
www.worlddyevariety.com
List of Suppliers: Direct Red F3,Direct Fast Red BA,Direct Fast Red F3B. Tianjin Yadong Group . ACDI Red 8 BLN(Aakash Chemicals & Dyestuffs Inc)Alacodirect Red 2BL(Classic Dyestuffs Inc)AmbidirectRed 3BL( Thai Ambica Chemicals Co Ltd) Anadurm Red D-BA( Albion Colours Ltd) Arid Red 8 BLN ( Aashiana Dyestuffs Inc) Best Direct Supra Red F3B( Oriental Giant Dyes and Chemical Ind Corp)
Other dyes with "sirius red" in their trivial names probably are not suitable if they are not the product recognized in the Colour Index as  CI 35780,  Direct red 80. An example of a different dye is sirius red 4B (= CI 28160 = Direct red 81), which has been prescribed for use in some staining techniques as a dye with properties similar to those of eosin Y and acid fuchsine.


The Biological Stain Commission has standards for sirius red F3B as a collagen stain. Dye from a batch that meets their standards will be OK for your method.


My Histonet post from which you are using this method must date from the 1990s, when I knew that picro-sirius red solutions were good for 5-6 years. (I would have written 3 years to be cautious.)  With more experience with stored and newly made solutions, I feel confident in saying they keep for more than 20 years.


It might get contaminated from too much iron-haematoxylin extracted from previously stained  slides. I don't know what this would do.


The most obvious cause of red cytoplasmic staining by picrosirus is not enough picric acid (yellow powder in the bottom of the bottle) in the staining solution.


It's unfortunate that items found with HistoSearch are undated. It doesn't matter in this case, but many Histonet items become outdated after only a year or two; antibodies and automated staining are examples of fields in which you need to know the age.

Keep in touch about your sirius red problem.

John Kiernan
John A. Kiernan MB, ChB, PhD, DSc
Professor Emeritus, Anatomy & Cell Biology
University of Western Ontario, London, Canada
    https://www.schulich.uwo.ca/anatomy/people/bios/emeriti/kiernan_john.html
Also  Secretary,  Biological Stain Commission, Inc.
    https://biologicalstaincommission.org
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________________________________
From: abi jag via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 09 August 2019 11:29
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] Recent issues with picro sirius red staining (entire liver section become red, no yellow background)

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.


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