Methods and Techniques Discussion => Immunocytochemistry (ICC) => Topic started by: Dunia on August 27, 2010, 07:12:50 AM

Title: preservation of 3D structure of cells growing in suspension
Post by: Dunia on August 27, 2010, 07:12:50 AM
Hello everyone!

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)

Thank you!
Title: Re: preservation of 3D structure of cells growing in suspension
Post by: Dunia on September 01, 2010, 04:29:58 AM
I’ve searched the answers to my questions and that is what I found:
Tips from Susan Anderson
That is what she says:
“When looking at B cells (they are relatively fragile) I have tried to support the coverslip with EM grids on two sides, 12  m diameter latex beads from Sigma (LB-120) mixed with the final cell suspension in antifade medium, and have tried #1 coverslips instead of #1.5. Using #1 coverslips has given me the fewest smashed cells. I now use an inverted scope and pipet the cells onto coverslip chamber slides (Lab-Tek #136439) in antifade medium so no worries about flattened, smashed cells.”

My other mistake was methanol as a fixative. Methanol was really good for getting perfect Golgi staining, but unfortunately: “Quantitative comparisons of different fixation protocols have shown that cultured cells fixed with cold methanol shrink by as much as 50%.” (from Cell Biology Applications of Fluorescence Microscopy by Stephen Rogers)
I found both this useful links on
Thank you!!!!

(any further comments would be really appreciated  :))