[Histonet] Masson Trichome Staining: Can I use Fast Green?

[John Kiernan]

"The slides have not been coming out well." is not very informative! Your problems may have nothing to do with which dye you are using as the collagen stain. There are plenty of other things that can make a multi-step trichrome method give unexpected results. 
 
Fast green FCF (C.I. 42053) is usually considered superior to light green SF (C.I. 42095) on account of being less prone to fading. Be sure you have the correct dye, and that it is from a batch certified by the Biological Stain Commission (B.S.C. - see the label on the bottle of dye powder, which will also tell you the dye content). If you buy a ready-made solution, check with the supplier. 
 
The minimum acceptable dye content for certification of fast green FCF (85%) is higher than the minimum for light green SF (65%). A 2% solution of the former might therefore have greater tinctorial power than a 2% solution of the latter. The B.S.C. tests both dyes as substitutes for aniline blue in Gomori's one-step trichrome method; the concentration of blue or green dye in the mixture is 0.3%, irrespective of the dye content of the powder.  Gomori's is a more stringent test for dyes than Masson's trichrome method because there is no visual/manual control of the staining. A dye that works with Gomori's method should work well with Masson's. 
 
The Masson variant that you have been trying (from Bryan Llewellyn's excellent web site,  http://stainsfile.info/StainsFile/stain/conektv/masson.htm) is well documented. Follow up some references from Bryan's citation of the Bancroft & Stevens book, and learn the reasons for all the 11 steps of the modified Masson technique. The current edition (6th, 2008) of "Theory and Practice" is edited by Bancroft & Gamble: ISBN 9780443102790.
 
My textbook (ISBN 97819048422) covers trichrome methods in Chapter 8, with plenty of references to scientific/scholarly literature, much of which is now available on the internet, especially if you can go through a university or public library's web interface. There is a well ilustrated chapter on troubleshooting trichromes, by Vinnie Della Speranza, in R.W.Brown's (2009) "Common Problems" book, ISBN 9780930304959 (pp. 95-101).
 
A rinse in tap water at any stage after after washing out the iron-haematoxylin nuclear stain can weaken either the red or the blue/green component of any trichrome method. So can "graded alcohols" for the final dehydration of well stained slides. 
 
John Kiernan
Anatomy, UWO
London, Canada
= = =
----- Original Message -----
From: cscampbe <@t> uci.edu
Date: Thursday, April 8, 2010 18:42
Subject: [Histonet] Masson Trichome Staining: Can I use Fast Green?
To: HistoNet <histonet <@t> lists.utsouthwestern.edu>

> Hi Histonet,
> 
> I am currently using the protocol from Stainsfile for staining heart
> tissue: http://stainsfile.info/StainsFile/stain/conektv/masson.htm
> 
> My lab had Fast Green readily available so I substituted it for Light
> Green in Solution C. The slides have not been coming out well. I 
> failed to
> account for the difference in timing between FG and LG - running 
> the slide
> in FG for the full 10 minutes. Has anyone used FG in Masson's 
> Trichrome?If so, how long did it take to get the desired colors 
> in the solution? Or,
> is Fast Green a poor substitute for Light Green?
> 
> Thanks for your advice!
> -Colin
> 
> 
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