[Histonet] Masson Trichrome

Thu Apr 16 19:14:06 CDT 2009

I'll send you our procedure under a separate cover.

Couple of thoughts:
1. Try extending time in Bouins to 1 hour. If you put room temperature
Bouins in the 60 degree oven with your slides, at 30 minutes, it's just
finally getting up to 60 degrees. So extend the time to 45-60 minutes, so it
has 15-30 minutes at 60 degrees.

2. Weigert's usually ends up being pulled out. Most of the time, the
pathologists don't even notice that the nuclei are red. They just care to
differentiate blue collagen from red muscle/cytoplasm.

3. There are a variety of red dyes that can be used, each one giving you a
slightly different shade and intensity of red. Most are interchangeable -
many of the Ponceau varieties can be used, as well as biebrich scarlet, acid
fuchsin, etc. Of the Ponceau, the Ponceau S (CI #27195) seems to be most
intense, but is a little more on the pinky/magenta side of red.

4. We use just PTA. PMA costs a lot more, and we didn't find any improvement
over plain ole PTA.

5. The Aniline blue is probably the most important step, and works best if
checked with the microscope. Some specimens become blue very quickly, and
look best if pulled out after 1-2 minutes. Some need 5 minutes. Some need 10
minutes. I've found, it the collagen doesn't stain by 10 minutes, it's not
going to get any bluer, and in fact the red muscle begins to pick up some of
the blue after 10 minutes. I also found, the more you wash in water to check
the intensity of blue, the more the red muscle picks up blue, and the blue
collagen begins to look purplish. So I check it at 2 minutes and 5 minutes,
and pull it out at 2 or 5, if it's the right intensity. If the blue is too
pale at 5 minutes, I leave it for the full 10 minutes, and then pull it out
at that point.

6. Cut down the time of water rinses after aniline blue to a couple of
changes, 5 seconds each. (see #5 for reason).

7. If you are having problems with pale blue intensity, skip the acetic acid
rinse completely. Make the stain more "delicate", but can be skipped.

Peggy A. Wenk, HTL(ASCP)SL
Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lucie
Guernsey
Sent: Thursday, April 16, 2009 6:07 PM
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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