Collagen coated scaffold and hoechst stain

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Collagen coated scaffold and hoechst stain

Postby Chriscbe » Jun 22 2010 8:07 pm

I have grown fibroblasts on a 3D scaffold that I coated with a thin layer of collagen to make them stick. I'd like to do a DNA stain but I'm worried about the collagen either absorbing some of the hoechst stain or impeding it's diffusion to some of the cells. Given this scenario, do you think I absolutely MUST dissolve the collagen coating? Or could I get away with just applying the stain?

Thanks for any responses
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Re: Collagen coated scaffold and hoechst stain

Postby CrowSan » Jun 23 2010 10:04 am

Other people in the literature appear to have used hoechst stains within collagen scaffolds with no trouble. There is no reason why the hoechst should bind to the collagen with any great avidity. After staining a few simple wash steps (i.e. 3x PBS, 0.1% tween washes) should remove any unbound dye. I have certainly used DAPI with collagen and collagen antibodies with no noticable background staining. If you are intending to use a plate reader though I highly recommend getting rid of any traces of DMEM as this really seems to interfere with hoechst and DAPI signals!
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