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

[Elizabeth Chlipala]

Krista

Not sure if this would help but we mordant in Bouins for 1 hour at 60C, we let the bouins come up to temp (60C) before we put the slides in.  We have not had good success with temps lower than 60C and if we do not warm up the bouins.  We also find that fresh reagents are key in successful results, we make up our reagents, we don't use a kit.  If you don't need the alcian blue portion of the stain present, my suggestion would be to try a modified trichome, - mordant in bouins and in place of the iron hematoxylin use verhoff elastic stain, differenciate and then counterstain with a regular trichrome.  This will turn out really nice and it will demonstrates elastic fibers, collagen and muscle nicely.  We prefer doing this over the Movats.  I have a protocol if you need one.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Laboratory Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
Work (303) 682-3949
Fax (303) 682-9060
Cell (303) 881-0763
liz <@t> premierlab.com
www.premierlab.com

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Krista Sider
Sent: Friday, May 10, 2013 3:13 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [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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