AW: [Histonet] Trichrome & Fixation

Fri Mar 6 11:08:50 CST 2015

Quite right! Treating sections of formaldehyde-fixed tissue with Bouin's fluid is not mordanting; binding site retrieval is probably a more accurate term. 

Picric acid induces a change in the section that improves the selective uptake of the heteropolyacid and the larger dye anions by collagen and of the smaller dye anions by cytoplasm. Picric acid alone is just as good as Bouin for this purpose. The picric acid treatment has the effect of making a section of formaldehyde-fixed tissue more like a section of tissue fixed in a picric acid-containing fixative such as Bouin or Gendre. For spectacular trichrome staining you need a fixative that contains mercuric chloride, like Zenker or SuSa.

An example of a mordant in biological staining is iron alum in Heidenhain's method. The subsequently applied haematein solution forms a coloured complex with the iron bound by the sections. The differentiation in iron alum solution then removes bound haematein by forming a water-soluble complex. The word "mordant" is probably best avoided for staining with pre-formed dye-metal complexes such as carmine, aluminium-haematein (haemalum), Weigert's iron-haematein and gallocyanine-chrome alum. See R.W.Horobin (1982) "Histochemistry" Stuttgart: Gustav Fischer (ISBN 3437107003 or 0407002480), page 77.

John Kiernan
= = =
On 06/03/15, Gudrun Lang  <gu.lang <@t> gmx.at> wrote:
> I disagree. Usually a dye binds covalently to the mordant, and a mordant should be a multi-valent metal (like aluminium, ferrum, molybden,..). The dye-mordant-complex binds via the mordant to the substrate.
> Picric acid of Bouin's is washed out before staining.
> 
> It may be a matter of definition of the term mordant.
> 
> Gudrun
> 
> -----Ursprüngliche Nachricht-----
> Von: Johnson, Carole [mailto:cjohnson <@t> nmda.nmsu.edu] <cjohnson <@t> nmda.nmsu.edu]> 
> Gesendet: Freitag, 06. März 2015 15:10
> An: gu.lang <@t> gmx.at
> Betreff: RE: [Histonet] Trichrome & Fixation
> 
> Boiuns solution acts as a mordant in trichrome stains
> 
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] <histonet-bounces <@t> lists.utsouthwestern.edu]> On Behalf Of Gudrun Lang
> Sent: Friday, March 06, 2015 1:13 AM
> To: 'Amos Brooks'
> Cc: histonet <@t> lists.utsouthwestern.edu
> Subject: AW: [Histonet] Trichrome & Fixation
> 
> Hi,
> years ago we did a stain called CAB (=one step trichrome) regularly on liver-tissue. I don't know if it was because of ignorance or with aim, but it was done without Bouin. The result was blue-grey hepatocytes and darker blue collagen. - also totally different to the result with Bouin (red hepatocytes).
> 
> I think the Bouin is less a "re-fixation" than more an "binding-site retrival" in this context.
> 
> Gudrun
> 
> 
> -----Ursprüngliche Nachricht-----
> Von: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] <histonet-bounces <@t> lists.utsouthwestern.edu]> Im Auftrag von Amos Brooks
> Gesendet: Donnerstag, 05. März 2015 22:01
> An: histonet <@t> lists.utsouthwestern.edu
> Betreff: [Histonet] Trichrome & Fixation
> 
> Hi,
>  It is interesting that you should mention the importance of fixation on the Trichrome stain. I have an image of two murine hearts processed, cut and stained side by side. The only difference between the two is that they were harvested at different times, so one sat in formalin long enough to be properly fixed the other one was placed in the fixative then immediately brought in to be processed from 70% ethanol on. They are *totally* different looking. The red muscle tissue looks more purple therefore less distinct from the blue blood vessels. You get a similar effect with lung bronchial epithelium.
> 
> Cheers,
> Amos
> 
> On Tue, Mar 3, 2015 at 1:00 PM, <histonet-request <@t> lists.utsouthwestern.edu>
> wrote:
> 
> > Message: 15
> > Date: Tue, 03 Mar 2015 12:34:33 -0500
> > From: John Kiernan <jkiernan <@t> uwo.ca>
> > Subject: Re: [Histonet] Masson trichrome and H and E
> > To: Emily Brown <talulahgosh <@t> gmail.com>,
> > "histonet <@t> lists.utsouthwestern.edu"
> > <histonet <@t> lists.utsouthwestern.edu>
> > Message-ID: <7390afa3112a.54f5aa59 <@t> uwo.ca>
> > Content-Type: text/plain; CHARSET=US-ASCII
> >
> > If you can't get two colours with H&E, don't expect to get the colour 
> > scheme right with Masson's trichrome, which needs more skill. If you 
> > are hoping to show basement membranes in the kidney, you would do 
> > better to use a technically simpler staining method such as 
> > picro-sirius red or periodic acid-Schiff. If for some reason you 
> > really need three colours, a one-step trichrome such as Gomori's, 
> > Cason's or Gabe's might be the way to go rather than Masson's or one 
> > of the other multi-step trichromes. Remember that all trichrome 
> > methods are greatly influenced by the fixative. A post-fixation 
> > treatment of the sections, usually with picric acid, is needed for 
> > formaldehyde-fixed tissues. Some alternative post-fixation treatments 
> > were proposed by Yu & Chapman (2003) J. Histotechnol. 26(2): 131-134, but their coloured photos were not very convincing.
> >
> > Making up staining solutions in-house is always cheaper than buying 
> > pre-made solutions.
> >
> > John Kiernan
> > London, Canada
> >
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