[Histonet] Masson Trichrome

[Tony Henwood]

A few comments that might be of help:

Allow the alcoholic haematoxylin to ripen for a few weeks prior to use
or if in a hurry use a celestine blue-Harris's Haematoxylin stain.
Use the smooth muscle cells in blood vessels to gauge the
differentiation step. My experience is to differentiate until a little
pink still remains in the collagen since the acidic blue or green
solution will continue to differentiate the red muscle stain.
The blue collagen solution stains faster than the green solutions so use
for a shorter time.


Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lucie
Guernsey
Sent: Friday, 17 April 2009 8:07 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Masson Trichrome


Hi Histonetters - I could really use your help! I recently received a
protocol for a Masson Trichrome stain that, if followed exactly, does
not seem to work. I've been doing a lot of online and publication
research, and yet I still have questions. I'm currently attempting to
stain 3 um thick PFA-fixed paraffin kidney sections (mouse and rat). The
following is my current protocol with my questions/problems in *bold*.
Thank you so much in advance - I'm pulling my hair out over this!

1. Standard deparaffinization/rehydration.
2. Bouin's solution for 30 min at 56-60 degrees C
3. Running tap water for 8 min (or until not yellow). Rinse with dH2O.
4. Weigert's iron hematoxylin for *1 hr (all protocols claim 5 min -
what am I doing wrong???? Do the stock solutions need to ripen before
use????)*
         - Solution A:  5 g Hematoxylin + 500 mL 95% EtOH
         - Solution B:  20 mL 30% aqueous Ferric Chloride + 500 mL dH2O
+ 5 mL HCl, concentrated
         - WORK solution: equal parts Solution A and Solution B - made
immediately before use - turns black 5. Running tap water for 5 min.
Rinse with dH2O. 6. Scarlet Acid Fuchsin for 5 min. *(I realize that
most protocols call for Biebrich Scarlet - are Biebrich or Xylidine
Ponceau* *necessary????)
*         - 0.5 g Acid Fuchsin + 0.5 mL Acetic Acid, glacial + 100 mL
dH2O
6. Rinse with dH2O.
7. 1% Phosphotungstic acid for 8 min *(most protocols call for a
phosphotungstic/phosphomolybdic acid mixture - is PMA necessary????)*
         - 1 g Phosphotungstic Acid + 100 mL dH2O
8. Aniline blue - *the time for this is what I'm struggling to determine
- have tried 5, 10, 15, 20, 25 min and all have been very light/pale -
will try even longer times, though most protocols suggest 5-10 min....*
         - 2.5 g Aniline Blue + 2.5 mL Acetic Acid, glacial + 100 mL
dH2O 9. Rinse with dH2O. 10. 1% Acetic acid for 1 min 30 sec *(I will
try decreasing to 1 min in an attempt to get my aniline blue to stay
darker)* 11. Rinse with dH2O. 12. Dehydrate: 95%, 100%, 100% EtOH, 30
sec. each. 13. Clear: Xylene, 3x, 3 min. each. 14. Mount using DPX
(salicylic balsam based mounting medium)


* While my hematoxylin works if I stain for an hour, I would love to
know how people are able to stain for only 5 min - when I try that, it
all just rinses out by the time I mount the slides....
* My scarlet acid works ok - light pink to reddish - but if I was to use
Beibrich or Ponceau, would it make it better/clearer?
* Is phosphomolybdic acid necessary for good differentiation? If so,
does anyone have a quality, but inexpensive PMA that they use and can
recommend?
* How long for aniline blue/acetic acid?
* I get quite a bit of purple - obviously a mix of red and blue - but is
it too much red and too much blue, or is it that blue hasn't replaced
all the red yet????
* How many times can you reuse the scarlet acid and aniline blue
solutions? The hematoxylin, phosphotungstic acid, and acetic acid
solutions are one-time use, correct?

Thank you in advance for all your suggestions!

Lucie Guernsey
University of California, San Diego
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