Dear Angela Fogg,
The staining method seems reasonable if the ingredients of your bought "Gomori trichrome" solution are the same as in Gomori's original method. You should check with the supplier and Gomori's paper. In your original Histonet post, you also wrote:
> Fixed in 10% formalin for 3 weeks, processed
> with formalin, alcoholic formalin, alcohols
> and Clear Rite II ...
Taken literally this means the specimens went from 10% formalin (= 4% formaldehyde) to 40% formaldehyde in water (formalin) and then into some formalin-alcohol mixture before being dehydrated in "alcohols". This isn't usually done. I don't know what Clear Rite II is - ? limonene, a mixture of hexanes & heptanes, or a glycol ether. There are many "xylene substitutes", mostly poorly described by the suppliers, and not all have the same miscibilities as xylene or toluene.
The Histonet Archives contain many sob-stories about trade-secret clearing agents. For research, can you justify using any commercial product of unknown composition? No peer-reviewed journal will publish results that cannot be replicated.
You also wrote:
> ... embedded in EM400 paraffin ...
Check the melting temperature of this product. Staining, especially by trichrome methods, is profoundly affected by the type of wax. An important paper about this was published by Allison & Bryant (1998) in Biotechnic & Histochemistry 73(3):128-136.
John Kiernan
Anatomy, UWO
London, Canada
= = =
----- Original Message -----
From: angelafogg@aol.com
Date: Monday, November 10, 2008 20:31
Subject: Re: [Histonet] Trichrome Question
To: jkiernan@uwo.ca
> The contents of this kit is as follows with the timing attached.
>
> Bouins for 1 hour at 60 degrees C
>
> Wash
>
> Gomori's One Step Trichrome for 15 minutes
>
> 1% Acetic acid for 1 min
>
> dehydate,clear etc
>
> Simple stain, easy to remember and works every time except for
> this one piece of colon.
>
> Thanks, glad you are interested in assisting my quest to get an
> answer.
> Regards,
>
> Angela
>
>
>
>
>
> -----Original Message-----
>
> From: John Kiernan
>
> To: ANGELAFOGG@aol.com
>
> Cc: Histonet@lists.utsouthwestern.edu
>
> Sent: Mon, 10 Nov 2008 2:05 am
>
> Subject: Re: [Histonet] Trichrome Question
>
>
>
>
>
>
Dear Angela Fogg,
>
>
>
> It is difficult to reply without knowing what's in the "Chromaview Stain kit Gomori's Trichrome." How does this differ from the method published by Gomori in Am. J. Clin. Path. 20: 662-664 (1950)?
>
>
>
> John Kiernan
>
> Anatomy, UWO
>
> London, Canada
>
> = = =
>
> ----- Original Message -----
>
> From: ANGELAFOGG@aol.com
>
> Date: Saturday, November 8, 2008 10:03
>
> Subject: Re: [Histonet] Trichrome Question
>
> To: jkiernan@uwo.ca
>
>
>
> >
>
> >
>
>
>
>
>
> >
>
> >
>
> >
>
>
>
>
>
>
>
> > Thanks for responding. Fixed in 10% formalin for 3 weeks, processed
>
> > with formalin, alcoholic formalin, alcohols and Clear Rite III for Richard
>
> > Allan, embedded in EM400 paraffin from Surgipath. Cut at 5 microns and stained
>
> > using Richard Allan's Chromaview Stain kit Gomori's Trichrome. Used the
>
> > above method on our routine Trichrome controls and all the muscle stained red as
>
> > they are supposed to be. That's why I put out the question. The blue
>
> > staining of the muscle in the colon puzzled me. All patient tissue has
>
> > stained correctly as well.
>
>
> > Thought maybe the length of time in formalin had something to do with the
>
> > blue staining. Maybe longer than an hour in Bouin's before continuing the
>
> > Trichrome would help?
>
>
>
> > Await your thoughts and info
>
>
>
> > Regards
>
>
>
> > Angela
>
>
>
> >
>
>
>
> >
>
> > In a message dated 11/8/2008 2:10:03 A.M. Eastern Standard Time,
>
> > jkiernan@uwo.ca writes:
>
>
>
> >
>
Which
>
> > trichrome method? What kind of section (frozen, paraffin, plastic?)
>
> > Any
>
> > post-fixation in something other than plain formalin before staining?
>
> >
>
> > Provide the technical details, and you will get lots of advice!
>
> >
>
> >
>
> > Weeks in formaldehyde is not "prolonged" fixation unless the
>
> > weeks
>
> > are numerous enough to make years. As a generality, trichrome
>
> > methods
>
> > do not work very well after fixation in liquids that have
>
> > formaldehyde as the
>
> > only active ingredient. Postfixation of the sections
>
> > can compensate. Bouin
>
> > is frequently used. Saturated aqueous picric acid us
>
> > just as good. There are
>
> > published reports that iodine and even citrate
>
> > buffer will improve trichrome
>
> > staining of paraffin sections of
>
> > formaldehyde-fixed tissue. See Yu &
>
> > Chapman 2003 J.
>
> > Histotechnol. 26(2): 131-134.
>
> >
>
> >
>
> > "Trichrome" has been applied to several staining
>
> > techniques that use two
>
> > or more dyes. By convention, since about 1920,
>
> > trichrome methods have
>
> > been those using phosphomolybdic or phosphotungstic
>
> > acid (or both) to
>
> > enable the staining of collagen and cytoplasm by
>
> > anionic dyes with sharply
>
> > contrasting colours: blue or green for
>
> > collagen, and red for cytoplasm
>
> > (including smooth & striated muscle). A
>
> > third anionic dye, typically yellow
>
> > or orange, may be added to stain red
>
> > blood cells. Instructions for
>
> > trichrome methods can be found in all
>
> > textbooks of microtechnique and
>
> > histotechnology.
>
> >
>
> > John
>
> > Kiernan
>
> > Anatomy, UWO
>
> > London, Canada
>
> > = = =
>
> > ----- Original Message
>
> > -----
>
> > From: angelafogg@aol.com
>
> > Date: Friday, November 7, 2008
>
> > 20:17
>
> > Subject: [Histonet] Trichrome Question
>
> > To:
>
> > histonet@lists.utsouthwestern.edu
>
> >
>
> > >
>
> > > Performed a trichrome
>
> > stain on a piece of colon which had been
>
> > > in formalin for? weeks.?
>
> > Muscle stained blue instead of
>
> > > red.?What happened?? Does prolonged
>
> > fixation react this way?
>
> > > Hope someone can shed some light on
>
> > this.
>
> > > Regards,
>
> > > Angela
>
> > >
>
> > _______________________________________________
>
> > > Histonet mailing
>
> > list
>
> > > Histonet@lists.utsouthwestern.edu
>
> > >
>
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
> >
>
>
>
> >
>
>
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