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

Fri Apr 9 11:26:44 CDT 2010

** Proprietary **
** Reply Requested When Convenient **

Colin,

If you havin difficulty with MT try the Gomori One Step 

1.  Sections to water.
2.  Pretreat in 3% Potassium dichromate for 1hr at 60ºC 
3.  Stain nuclei with celestin blue/Mayer's haematoxylin. 5  min
4.  Wash in tap water.
5.  Differentiate and blue.
6.  Stain with chromotrope green mixture      --- 10 mins.
7.  Rinse in distilled water
8.  Blot and dehydrate from absolute alcohol ,clear and mount. 

Results
Muscle and fibrin	-  Red
RBC's 	                -  Red
Collagen       	-  Green

MASSON TRICHROME (ONE-STEP)
Ingredients:
Chromotrope 2R 		0.6 g.
Fast Green FCF 		0.3 g.
Phosphotungstic acid 	0.6 g.
Glacial acetic acid 		1 ml.
Distilled water 		100 ml.

METHOD:-
Dissolve the chromotrope 2R, the fast green and the phosphotungstic
acid in the water and add the glacial acetic acid.

Hope this help

Malika Benatti

Specialist BMS 
Camelia Botnar Laboratories
Histopathology Department
Great Ormond Street Hospital
London WC1N 3JH

Tel:  +44 20 7405 9200 ext 5475
Fax:  +44 20 7829 7875

benatm <@t> gosh.nhs.uk

>>> John Kiernan <jkiernan <@t> uwo.ca> 09/04/2010 17:08 >>>
"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
> 
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu 
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu 
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

*********************************************************************************************************
This message may contain confidential information. If you are not the
intended recipient please inform the sender that you have received the
message in error before deleting it.

Please do not disclose, copy or distribute information in this e-mail or
take any action in reliance on its contents: to do so is strictly
prohibited and may be unlawful.

Thank you for your co-operation.

*********************************************************************************************************