[Histonet] Recent issues with picro sirius red staining (entire liver section become red, no yellow background)
Kate Davoli
kdavoli at gmail.com
Mon Aug 12 10:59:06 CDT 2019
I suspect the "all red" as opposed to "only some red" is because the PSRed
is not de-staining out of the non-collagen structures. My guess is that
your Solution B is old. Acetic acid becomes weak in water very fast so I
make the 1% acetic acid solution fresh every time and use the same day.
Hope this helps!
On Mon, Aug 12, 2019 at 7:44 AM Dr. Michael Gudo (Morphisto GmbH) via
Histonet <histonet at lists.utsouthwestern.edu> wrote:
> The sirus-red stain is a stain to differentiate collagene I against
> collagene III in polarized llight, so in bright field all fibres will be
> red, just if you change to polarised light, you can differentiate both
> kinds of fibres by their birefrigence which is orange to yellow for
> collagene 1 and green for the collagene 3.
>
> Cheers
> Michael
>
>
> > Am 10.08.2019 um 06:29 schrieb John Kiernan via Histonet <
> histonet at lists.utsouthwestern.edu>:
> >
> > 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
> > = = =
> > ________________________________
> > 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
> >
> > |
> > |
> > | |
> > Sirius red collagen procedure
> >
> >
> > |
> >
> > |
> >
> > |
> >
> >
> >
> >
> > 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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>
>
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