AW: [Histonet] Movat Pentachrome - Can't Remove Woodstain Scarlet-Acid Fuchsin from Collagen

[Gudrun Lang]

Try to stain first in PTA/PMA solution to impregnate the collagen fibers -
perhaps testing with different times. Then follow up with red stain and the
usual procedure.

We use a stain called SFOG, that first impregnates 2 min with PMA and
afterwards with the mixture of Chromotrop, Acidfuchsin and Anilinblue for 10
min. The longer we do the Polyacid-step the more intensive are the fibres
and less intensive is the cytoplasma.
I think, if after this trial the collagen is still red, that there are
binding-sites in the collagen, that can't be influenced. Maybe acidfuchsin
is here the main partner and a pure solution of Scarlet Red may help.

Gudrun Lang

-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Krista
Sider
Gesendet: Freitag, 10. Mai 2013 23:13
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] Movat Pentachrome - Can’t Remove Woodstain Scarlet-Acid
Fuchsin from Collagen

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
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