[Histonet] Re: Masson Trichrome

[Lucie Guernsey]

Thank you all for your suggestions! Though, if anyone still has ideas,
please don't consider this thank you as a closing of the subject - I (and
I'm sure, others who are in my situation) would love to hear any and all
variations that work. Now I'm off to cut more tissue so I can run more
stains using your suggestions and, cross your fingers for me, hopefully
(finally) optimize this stain for my lab....

Thanks again!

Lucie Guernsey
University of California, San Diego



On Thu, Apr 16, 2009 at 3:07 PM, Lucie Guernsey
<lucie.s.guernsey <@t> gmail.com>wrote:

> 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
>