[Histonet] Movat Pentachrome - Can’t Remove Woodstain Scarlet-Acid Fuchsin from Collagen

[Krista Sider]

Hello All,

I have successfully stained porcine and mouse paraffin embedded heart
tissues with Movat’s Pentachrome (MP), using EMS’ MP solutions and protocol
(http://www.emsdiasum.com/microscopy/technical/datasheet/26385.aspx), with
some optimization on times.

However, now I am working with archival human tissue that has been fixed
for much longer and is much older than my other samples (Human Aorta &
Aortic Valve, formalin fixed 2-6 months or 2-4 years, embedded in paraffin
and stored at RT for ~6 years). I cannot seem to get the Woodstain
Scarlet-Acid Fuchsin (WSAF) to pull out of the collagen with the 5%
Phosphotungstic Acid (PA) or 1% Phosphomolybdic acid (PM) Acid. I have
tried increasing the Bouin’s initial fixation (max 2 hrs 50*C), diluting
the standard WSAF solution by 50% (1 dip) and taking the PA or PM up to 1
hr, yet there is always a lot of red still left in the collagen, in some
regions as strong as the muscle (which I am sure are not muscle or dense
cells). I see virtually no change with increased PA or PM time. As I have
aorta in my samples I can’t just leave out the muscle stain.

I would be very grateful for your insights into anything I could try to get
clean collagen and stained muscle in the MP stain. Why might my method not
be working? Is there something I can substitute for the WSAF that might be
appropriate?

Thank you very much for you help,

Krista