We need to perform a confocal microscopy on a cell culture growing in suspension to colocalize our protein of interest with ER or Golgi.
I prepared cell smears by air drying cells in 20% FCS on slides, fixed with ice cold methanol, and preceded to double immunofluorescent staining (very standard procedure). The Abs stained the proteins OK, but there was no complete overlapping with either Golgi or ER markers. The pattern of staining may be described as mostly ER, very rarely Golgi, some in non of the compartments. When we follow the same procedure with a different cell line that are normally growing attached to the slides and have a flat stretched morphology, we stain them directly on the slides they grow on and get a much better pattern: complete colocalization with ER, non in Golgi, no leakage to other parts of the cell.
So we suspect that the pattern we get when staining suspension cells is an artifact due to the cells flattening during airdrying (the cell shaped as a ball becomes a pancake).
Could you please suggest me a way to preserve ball shape of the cells growing in suspension during staining, at the end they should be stuck to the slide to perform a confocal.
May be I should mount the cells in paraffin and stain cell sections? may be cytospin in some sort of medium? (I've never done non of the things I have suggested, sorry if they sound foolish)