Does anybody have a protocol for staining skeletal muscle using Procion Orange to look at membrane damage? We routinely use evans blue dye, but want to look at some alternative methods as well.
Thanks
Dear Mayank Verma
I advise you to read this publication :
Eur J Clin Invest. 2002 Jun;32(6):443-7.
Procion orange tracer dye technique vs. identification of intrafibrillar fibronectin in the assessment of sarcolemmal damage.
Palacio J, Gāldiz JB, Alvarez FJ, Orozco-Levi M, Lloreta J, Gea J.
Source
Department of Pneumology and Pathology, Hospital Del Mar - IMIM, Universitat Pompeu Fabra, Barcelona, Spain.
Best regards
Fabio
Dear Mayank Verma,
another article, perhaps also of interest to you could be:
http://php.scripts.psu.edu/staff/e/b/ebl3/eblimages/NatureMed.pdf
Greelish et al, 1999: Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector
(using Procion Orange, and some hint on Evans blue, interesting topic and images...). Also there exists another, old reference, Moens P., Baatsen PHWW, Marechal G. Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch. J.Muscle Res Cell Motil.14, 446-451,1993 using Procion red as bathing solution for demonstration of sarcolemmal damage. Best wishes and good luck, Wolfgang
Dear Susan,
I didn't find any valuable information in the URL you posted, dealing with the requested matter (sarcolemmal membrane damage localization):
the list retrievable as pdf consists of several dye combinations (with ProcionMX) to achieve special color tones.
The basic information on (used in that particular application more/less as cloth fibre stain) can be found on
http://www.jacquardproducts.com/procion-mx.html
and instructions for use are available @http://www.jacquardproducts.com/assets/jacquard-site/product-pages/dyes/procion-mx/Procion%20MX%20Instructions.pdf.
One for sure could try whether the coloring dye intended for fabrics or used as dye for artists is suited for the task of localizing sarcolemmal (muscle fibre) membrane defects, but there is not any hint on such incorporated in the information leaflets on JACQUARD's website (http://www.jacquardproducts.com/company/) . Disclaimer: no financial interest, no affiliation to that company.
Regards,
Wolfgang
One of my colleagues gave me the following protocol.
Procion Orange Staining
Solution
1% POD in PBS or Kreb Ringer buffer
Isolated muscle Protocol
1. Perform eccentric contraction in isolated muscle.
2. Incubate muscle is 1% POD in Kreb Ringer solution for 15 minute.
3. Freeze sample in liquid Nitrogen chilled isopentane in OCT
4. Cryosection at 10um.
5. Visualize at 580nm.
Infusion Protocol
100ul of 1% procion Orange in PBS can be injected IV by tail vein. The staining can be visualized between 1hr and 5 days. The paper decided to use 24 hour as their time point. (Covert persistence of mdx mouse myopathy is revealed by acute and chronic effects of irradiation.1999)
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