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

[Krista Sider]

Thank you all very much for you help. I really appreciate it.

Gudrun: I tried the PTA before the WSAF and it worked beautifully (1 min
PTA, dH2O, 5 min WSAF, dH20, AA, 10 min PTA). Red muscle and clean
collagen.Thank-you

Liz: I didn't get to trying the 60*C Bouin's but I will make a note for
next time I am optimizing.

Thanks again
Krista


On Sat, May 11, 2013 at 4:04 AM, Gudrun Lang <gu.lang <@t> gmx.at> wrote:

> 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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