Actin staining - (Feb/13/2012 )

Hi experts,

I am new in cell biology field so i in scream of helps from all experts.
I need to visualize cells' actin structure and distribution for cell cultured in different substrates, ie, glass, polystyrene, silicone polymers, and gel.
so i fix the cells in 4% paraformaldehyde for 45 min at RT, then PBS wash 3 times, then use phalloidin stain for actin overnight followed by 4 times washing with PBS.

But I couldn't see anything on my substrates T.T not only can't see any stained cells, but I couldn't see any cells

I did look at my cells before I fix them, so I am pretty sure there were cells on my substrates.

Anyone has any thoughts on it? Did I wash off my cells with PBS?

help....!! and thanks for any advices

You may well have washed the cells off. Which cell line are you using?

When you wash you need to be careful not to pipette directly onto the cells, you should run the PBS (or any other solution) down the side of the plate/well/flask, so that the solution covers the wells gently.