Hi there.. I have counted the cells manually in the fields collected from microscopy using the manual counting tool in ImageJ. As you probably harvest these cultures for IHC I have got good experience for looking at an IHC marker of your invading cells that does not show up in your fibroblasts that you use to make the organotypic matrix. In my case p53 was a good marker as I am studying mutant p53 which is highly expressed in my cells that invade and only very low expressed in the fibroblasts. It is even possible to have ImageJ count the 'brown' staining automatically. Alternatively I have made GFP-expressing cells and do the IHC with a GFP antibody which works as well. Unfortunately, because of the autofluorescence of collagen it is not very nice to work with GFP or RFP fluorescence to detect these cells in these matrices.
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