Why not just fix the frozen in Bouins, that is the fixative of choice for
Masson trichrome, save time and eliminate NBF altogether.  Fixation times
of 5, 10, 15 or even 30 min could be used to optimize staining/time.  Make
sure Bouins is made properly, with saturated picric acid.  This is
important for both its use for fixation and postfixation.

You did not say how long you were in the Biebrich scarlet/acid fuchsin and
the phosphotungstic acid/Phosphomolybdic acid step afterwards???  

One protocol in my file for decalcified bone sections, points out that the
connective tissue should be differentiated to colorless with the PTAH/PMA
using a microscope, and could it be you have done that PLUS removed too
much from the muscle fibers?

Just some thoughts

  


Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303