Hi Folks,

The following method is an alternative to the standard Masson technique and
has the advantage that it can be carried out on formalin fixed tissues
without the need to post chrome or fix in a mercury containing fixative
etc.  It is a method which was passed on to me in the 60's by my senior at
the time and who trained me in histological techniques.  I have no idea as
to the origins of the technique, perhaps John can come up with the answer
to this, but it is the method I prefer to the Massons and it worked
extremely well every time for me.  Hopefully some of you will give it a try
and I would be grateful for any feedback.

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Masson trichrome using a yellow mordant.

Fixation - routine fixatives containing mercury are recommended but formol
saline is satisfactory however fresh material is essential.

Solutions:-
1. Celestine blue and Mayer's haemalum for nuclear staining.

2. Yellow mordant- 
Stock solution 1 - 100 ml saturated solution of picric acid in 70% alcohol
plus 0.4g of orange G.  Dissolve the orange G in a small amount of
distilled water and add to the picric acid solution.
Stock solution 2 - 0.4g of lissamine fast yellow in 40 ml distilled water.
Add solution 1 to 2 and store at room temperature.

3. Ponceau 2R (ponceau de xylidine) 2g, plus 1g acid fuchsin, plus 200 ml
distilled water plus 3 ml acetic acid.

4. 1% phosphomolybdic acid.

5. 1% acetic acid

6. Connective tissue counterstain - 1% light green or methyl blue in 1%
acetic acid (1% direct sky blue in 1% acetic acid is another alternative)

Technique:-
1. Take 6 micron sections to water via the iodine/ sodium thiosulphate
sequence if necessary.
2. Stain nuclei in filtered celestine blue for 10 minutes, rinse in tap
water then 10 minutes in filtered Mayer's haemalum followed by a few
minutes in running tap water.
3. Stain in a working solution of the yellow mordant for 3 to 5 minutes
     (ie working solution is 30 ml of the combined solutions 1 and 2 in 70
ml of 80% alcohol).
4. Wash in running tap water until the yellow colour has been extracted
from the muscle - approx 1 minute.
5. Stain in the ponceau acid fuchsin solution for 5 minutes.
6. Rinse off the excess dye in tap water for approx. 20 seconds.
7. Place sections in 1% phophomolybdic acid until the red colouration is
retained in the muscle only and can be up to 30 minutes depending on the
freshness of the tissue and the fixation.
8. Place in the connective tissue stain (fresh material which has had 30
minutes in the phophomolybdic acid give 2 minutes, other material not so
well fixed which has had only 1 to 5 minutes in the phophomolybdic acid
give 45 seconds).  5 minutes for the direct sky blue irrespective of the
time in phophomolybdic acid.
9. Rinse well in 1% acetic acid followed by 75% alcohol.
10. Dehydrate clear and mount.

Results:_
Nuclei - blue/black
Muscle - red
Elastic tissue - pale pink
RBC's - yellow
Collagen, reticulin and mucin - green or blue depending on counterstain used.


Jim Hall,
MDA Equipment Evaluator,
Department of Histopathology,
University College London Hospitals,
Rockefeller Building,
University Street,
London, WC1E 6JJ.
Tel:  020 7679 6042
Fax:  020 7387 3674
Web Site: http://www.ucl.ac.uk/~rmkdhjh/index.htm