Hi, 

What is the scale of this picture ?It is likely that these spots are your Fibronectin. ECM prot like collagen often lead to spot-like signbals like these ones and hydrogels often lead to background despite washing toroughly between staining protocol steps. Maybe you can try 2 different concentrations of Fn (coating ?) and check the different intensity of staining between samples.

Hi Hadi,

I agree with Guillaume, although I have never incorporated fibronectin into a gel.  I have always stained for fibronectin on sections, or cells in culture.  In both cases, the signal can be punctate because the fibroblasts deposit it at their focal contacts, although in culture it can be fibrillar depending on how much the cell has secreted.  So I like Guillaume's idea for incorporating different amounts of fibronectin into your gels to see a change in signal intensity.  How did you incorporate the fibronectin into the gel?

Best,

Jill

I've also done some IF experiments against fibronectin and like Guillaume and Jill, have seen this punctate staining. I think this is likely genuine fibronectin staining, but Guillaume's idea is a great way to test this and know for sure.  I've also seen fibrillar staining, like Jill. 

Is this just a hydrogel without any cells? Fibronectin in serum exists as a soluble folded-up dimer, and requires cells with specific receptors (e.g. integrin alpha 5 beta1) to apply tension and build these dimers into a fibrous matrix. If your gel does not have any cells present, I wouldn't expect your fibronectin to exist as a matrix. 

There are protocols to have cultured fibroblasts deposit an intact matrix on various surfaces (and to strip away the fibroblasts when you're done, leaving only the matrix), or to even carefully collect these intact matrices for deposition elsewhere. If you're interested in that let me know and I'll try to dig up some of the literature I have on that procedure. 

fibronectin is used to coating coverslips and TC dishes for cell culture. it is wroted that it should diute carefully without any agitation in PBS without Ca, Mg salts. the picture looks like as an aggregated FN spots... i would expect some uneven staining but this is not look alike covering of a matrix surface. check your FN solution on a small glass coverslip, it is easy to find a protocol for that.

Thanks all.