Nuclei staining background in collagen matrix - (Feb/17/2012 )

Hello everyone,
I'm staining the nuclei of fibroblasts embedded in 3D collagen matrix using either dapi or PI. While the nuclei staining is clear, I always get high background (haze everywhere, sometimes isolated bright spots) in the matrix that are apparently not cells. Possible theories are non-specific binding of dapi to collagen fiber or DNA debris from dead and disintegrated cells. I tried 10% BSA blocking or DNase pre-treatment before permeabilizing cells, but none of them reduced the background. Does anyone know what this background is? precipitation of the dye or dapi is just too hard to wash out in a matrix?

BSA won't work - that's a protein block, not DNA. I would hazard a guess that the bright spots are areas where the DAPI/PI hasn't diffused all that well and has aggregated somehow.

Have you ever looked at unstained matrix? - perhaps the background is actually a property of the matrix (this is quite common for many things), rather than the staining process.