[Histonet] Masson's trichrome

[James Watson]

I have done the Masson Trichrome on human and all kinds of animal tissue with success, I follow the protocol in the AFIP "Laboratory methods in Histotechnology" with a couple of modifications:

Deparaffinize and hydrate to distilled water
Bouins overnight or 60 degrees for 1 hour (If heated seal staining jar and open in hood after cooling)
Wash in running water until sections are clear
Weigerts hematoxylin 10 min.
Running water 10 min
Rinse in distilled water
Biebrich Scarlet-acid fuchsin 15 min
Rinse in distilled water
Differentiate in Phosphotungstic-phosphomolybdic acid for 10-15 minutes (check under microscope if collagen tissue is clear, if not differentiate for 5-10 more minutes until collagen is clear.)
Counterstain in aniline blue for 2 minutes (or light green 10 min.)
Rinse in distilled water
Dehydrate, clear and mount.

Checking the differentiation is very important to ensure that all the red in not removed from the muscle and the collagen is clear and can pick up the counterstain.  Shortening the aniline blue time decreases the possibility of overstaining and eliminates the need for differentiating in acetic water which can also remove your hematoxylin.

Hope this helps.

James Watson HT, ASCP
Facilities Manager of Histology
GNF, Genomics Institute of the Novartis Research Foundation
Room C015
858-332-4647
jwatson <@t> gnf.org 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of NIDAL E MUVARAK
Sent: Wednesday, May 04, 2005 12:07 PM
To: Julien LambreyDeSouza
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Masson's trichrome


I also did MTS for the first time on human saphenous vein. Got intense 
blue staining throughout the vessel and some red in the middle, 
but no nuclear staining. Here's the protocol I followed (if 
you're interested, Julien):

Overnight in Bouin's solution
Wash in tap water for 1 min
Weigert's hematoxylin for 10 min
Wash in tap water for 1 min, blue in 37mM ammonia water Beibrich scarlet for 5 min Wash in distilled water for 1 min Phosphotungstic/phosphomolybdic acid for 10 min Discard and directly add aniline blue for 5 minutes Wash in distilled water for 1 min Wash in acetic acid for 1 min, then again with distilled water for 1 
min
Dehydrate, clear, coverslip

Anything wrong with this protocol? 


----- Original Message -----
From: Julien LambreyDeSouza <julien_lambreydesouza <@t> uqar.qc.ca>
Date: Wednesday, May 4, 2005 12:30 pm
Subject: [Histonet] Masson's trichrome

> 
> Hello,
> 
> I am trying out Masson's trichrome for the first time. I used the
> Goldner 
> modification from Gabe 1976, with Fuschine-Ponceau, orang-G 
> phosphomolybdic 
> and Light green. We are staining 7 micron sections of entire fish 
> larvae 
> heads. When following the protocol (5 minutes in each color 
> followed by a 
> 1% acetic water rinse) I get a general green staining of all 
> tissues. The 
> only way to differentiate cartilage from the rest in our section 
> is by 
> recognizing cellular topography, same as with Hematox-eosine 
> staining, 
> therefore loosing the whole point in using Masson's trichrome. I 
> seem to be 
> doing something wrong.
> 
> Would anybody familiar with the technique be inclined to share
> their 
> expertise or protocol? I would be happy to send a pic of our 
> staining 
> result by email to have input on the possible problem.
> 
> Thanks,
> 
> Julien.
> Julien Lambrey de Souza
> Assistant de recherche, Biologiste M.Sc.
> Biologie Évolutive.
> Université du Québec à Rimouski
> Département de Biologie, Chimie et Sciences santé
> 300, allée des Ursulines
> Rimouski (Québec) Canada G5L 3A1
> Tél.: (418) 723-1986 ext. 1714
> Fax.: (418) 724-1849
> Courriel: julien_lambreydesouza <@t> uqar.qc.ca
> 
> _______________________________________________
> 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