[Histonet] Re: Picro sirius red, fast green connective tissue stain

Tue Mar 8 02:36:45 CST 2011

Hi, John

The method is a two color staining method that stains collagen in red and
muscle fibers in green.
The filtration is in order to make the solution that is being used multiple
times and stored for a long period, more clear.
I've attached an article with an example (Fig2), that is done in the exact
way as i described before.
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9T-4T3KTBS-1&_user=626711&_coverDate=11/30/2008&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_acct=C000032999&_version=1&_urlVersion=0&_userid=626711&md5=805ac6cd7adeb0d8284a07f0be44dcb8&searchtype=a
<http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9T-4T3KTBS-1&_user=626711&_coverDate=11/30/2008&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_acct=C000032999&_version=1&_urlVersion=0&_userid=626711&md5=805ac6cd7adeb0d8284a07f0be44dcb8&searchtype=a>

Itai

2011/3/8 John Kiernan <jkiernan <@t> uwo.ca>

> Dear Itai Moshe,
>
> Can you explain the staining method you described in some Histonet
> correspondence nearly a year ago? The correspondence is in the "tail" of
> this email.
>
> Is it published? What is it used for? What is coloured yellow, green or
> red? Your procedure stops with a water rinse; are the slides then
> dehydrated, cleared and coverslipped?  Why does the picric-fast green
> solution have to be filtered twice?
>
> I ask these things because the Biological Stain Commission will soon be
> offering certification services for *sirius red F3B* (paper by R. Dapson
> et al., *Biotechnic & Histochemistry*, accepted Feb 2011). The BSC testing
> includes physico-chemical characterization of the dye,  picro-sirius
> staining for collagen, and an alkaline SR method for amyloid. Certification
> ensures the availability of dye batches that will work properly in their
> major applications.
>
> We are always interested in other uses of biological stains, which might
> become more important in the future.
>
> The method you describe is interesting in that the time in picro-sirius is
> very short (5 min). The most thorough mechanistic study of picro-sirius
> (Junqueira et al 1979 *Histochem. J.* 11: 447-455) found that the uptake
> of the dye by collagen fibres was quite slow, and recommended the
> now-standard 60 min. I have not tried the method you describe. I would
> expect it to stain the thickest collagen fibres red, erythrocytes yellow,
> and everything else, including thin collagen fibres and basement
> membranes, yellowy-green. Am I right?
>
> Any information you can provide about your picro-fast green + picro-sirius
> red method will be greatly appreciated, especially the evolution and actual
> value of the method.  Feel free to share this message with others who use
> the method.
>
> Yours sincerely,
>
> John Kiernan
>
> Secretary, Biological Stain Commission
>     http://biostain.com
> J.A.Kiernan  MB,ChB,PhD,DSc
> Professor, Dept of Anatomy & Cell Biology
> University of Western Ontario
> London,  Canada  N6A 5C1
>    http://publish.uwo.ca/~jkiernan/index.htm
> = = =
> ----- Original Message -----
> From: Itai Moshe <itai.moshe <@t> mail.huji.ac.il>
> Date: Wednesday, April 7, 2010 9:47
> Subject: [Histonet] picro sirius red, sirius red connective tissue
> stain[Scanned]
> To: histonet <@t> lists.utsouthwestern.edu, baza0013 <@t> d.umn.edu,
> David.Rushworth <@t> mail.bhrv.nwest.nhs.uk
>
> > picro Sirius red and Fast green protocol:
> >
> > Sirius red (Sirius Red F3B)- 0.1% in Picric acid.
> > Fast Green - 0.03% in Picric acid (100%).
> > Can be re-used, filter twice before each use.
> >
> >
> > 1) Deparaffinization and Rehydration:
> > A) Xylen - 10 min X2
> > B) Ethanol 100% - 2 Min X2
> > C) Ethanol 96% - 2 Min X1
> > D) Ethanol 80% - 2 Min X1
> > E) Ethanol 70% - 2 Min X1
> >
> > 2) Rinse in double distilled water (DDW) - 5 Min
> >
> > 3) Rinse in filtered Fast green - 20 Min
> >
> > 4) Wash with DDW ( just fill the slide box with DDW and empty).
> >
> > 5) Rinse in filtered Sirius red - 5 Min.
> >
> > 6) Wash with DDW ( just fill the slides box with DDW and empty).
> >
> > 7) Check in microscope, if the colors are not strong enough,
> > repeat from
> > step 2 to 7.
> >
> > That's it.
> >
> > --
> > Itai Moshe
> > Mark Pines lab
> > Institute of Animal Sciences,Volcani Center.
> > Dept. of Animal Sciences, School of Veterinary Medicine, The Hebrew
> > University of Jerusalem
> > Israel
> >
> > ------Original Message-----
> > *Adam Bazama* baza0013 <@t> d.umn.edu
> > <histonet%
> 40lists.utsouthwestern.edu?Subject=%5BHistonet%5D%20sirius%20red%20connective%20tissue%20stain%5BScanned%5D&In-Reply-To=
> >
> > *Mon Oct 6 10:27:37 CDT 2008*
> >
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> >
> > ------------------------------
> >
> > Hi Dave,
> >
> >
> >
> > I noticed that you posted a request in march of 2004 on histonet
> > for a
> > Sirius red fast green staining protocol. If you do have a
> > protocol, would
> > you mind sending it to me. I have had a hard time finding one to
> > compare to
> > mine and my lack luster results.
> >
> >
> >
> > Thank you,
> >
> >
> >
> >
> >
> > Adam Bazama, B.S.
> >
> > Junior Scientist
> >
> > Lillehei Heart Institute Histology and Microscopy Core Facility
> >
> > University of Minnesota Medical School, Division of Cardiology
> > 4-266 Nils Hasselmo Hall
> > 312 Church Street SE
> > Minneapolis, MN  55455
> > Lab Desk: 612-625-6779
> > Cell: 952-334-0607
> >
> > --------------------------------------------
> >
> > *David Rushworth* David.Rushworth <@t> mail.bhrv.nwest.nhs.uk
> > <histonet%
> 40lists.utsouthwestern.edu?Subject=%5BHistonet%5D%20sirius%20red%20connective%20tissue%20stain%5BScanned%5D&In-Reply-To=
> >
> > *Thu Mar 4 09:00:18 CST 2004*
> >
> >
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> >
> >
> > ------------------------------
> >
> > Can someone please supply method for Sirius red-fast green stain for
> > connective tissue.
> > Thanks in anticipation.
> > Dave.
> >
> >
> >
> > ------------------------------
> >
> >
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-- 
Itai Moshe
Mark Pines lab
Institute of Animal Sciences, Volcani Center.
Dept. of Animal Sciences, School of Veterinary Medicine, The Hebrew
University of Jerusalem
Israel