I've been working with murine neural stem cells as a 2d monolayer which is achieved by adding laminin (ECM) directly to the culture media, without the need to coat the TC plastics.

However, I've been having issues trying to set up experiments to perform immunofluorescence either on glass coverslips or poly-lysine coated chamber slides. I find that when I seed the cells as normal, the next day they form neurospheres and the monolayer is lost.

I've tried coating the coverslips/slides with culture media containing laminin at higher concentrations, which didn't seem to work. I've also tried coating with poly-l-lysine before adding an extra laminin coating and while this did help with the adherence and monolayer formation, the morphology of the cells appeared to be distorted.

I've tried various seeding densities too and I usually seed them so I can do treatments and fix/stain the following day....perhaps they need longer to get used to the surface of the glass? I guess another option I could try is to use plastic coverslips?

Has anyone performed ICC with these type of cells? I'd be grateful if anyone could advise me on any methods that would help with maintaining the monolayer.

Thanks in advance

David