[Histonet] Masson Trichrome

[Lucie Guernsey]

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