Cells or tissue slices are grown in 3D collagen matrices to more accurately replicate the in vivo cellular milieu that cells function in to study how cell morphology and function respond to the extracellular environment confining the cell. To faithfully capture the integrity and structure of the F-actin cytoskeleton (and any changes it undergoes) with a fluorescent phalloidin stain, proper fixation and handling of the cells/slices is necessary. For fixation of cells for phalloidin staining, methanol must be avoided and instead, paraformaldehyde or glutaraldehyde should be used as they provide excellent actin filament staining and good lamellipodia preservation.
Reagents
Equipment
Method
Phalloidin Staining Protocol
Prepare collagen-coated polyacrylamide gel substrate for cells
3D Culture Method
For detailed information on these reagents and methods, please see
Reagents and Equipment:
Same as Protocol 3 above with the following additions/exceptions
Method:
Phalloidin Staining Protocol:
For detailed information on these reagents and methods, please see: