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Next Subject 		Collagen stainingn@@n!2008-05-31Click here to register.
Can anyone suggest the available methods for staining and also quantification of collagen deposition in tissue.

Thank you
 
Re: Collagen staining
seanpeel2008-06-03Click here to register.
There are several methods you can use depending on what equipment you have.

You can measure the amount of hydroxyproline in the cell layer by HPLC or by colorimetric assay

(i.e.
Hydroxyproline measurement by HPLC: improved method of total collagen determination in meat samples.
Vázquez-Ortíz, et al.
Journal of Liquid Chromatography & Related Technologies, 2004 (Vol. 27) (No. 17) 2771-2780

Abstract:
A sensitive and selective assay of hydroxyproline is presented for the determination of collagen in meat and meat products. The amino acid is converted to a sensitive fluorescent derivative with 7-chloro-4-nitrobenzofurazan (NBD-Cl), followed by reversed-phased chromatography and fluorescence measurement (ex=465 nm, fl=535 nm). Hydroxyproline and proline can be measured by this system. The column and detector performance shows good retention, selectivity, and efficiency. Based on replicated analysis of controls over a range of 0-20 mg/ml, the method is accurate (94-100%) and precise (%CV of 0.3-13). The limit of quantification for hydroxyproline and proline was 0.0027 ng/ml. Fourteen meat samples with different levels of collagen normally found in meat and meat products were tested. The results showed that the linear range of assay was suitable for routine analysis, and applicable to the entire range of hydroxyproline levels normally found in meat and meat products (0.05-12.5%).

You can stain the cell layer with a colorimetric dye, that binds to collagen. After washing you elute the dye and then measure the absorbance on a plate reader.
You can make up the reagents yourself
(see the Sirus red assay http://meeting.chestjournal.org/cgi/content/abstract/130/4/230S-a) or there is a kit called the Sircol assay I have used that can be purchased.
see http://www.biocolor.co.uk/sircol.html

I have done both methods, the OH-proline method is the more common one, but requires more specialized equipment and is technically more demanding.
Neither are convienient, but both work.

There are also immunoassays for measuring the amount of collagen in solution, I am not sure if they would work on deposited collagen as you would have to solubilize it somehow.

Sean

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