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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet