Improving Membrane Staining of Cultured Cells Using Ferrocyanide as a Post-Fixative

Extract

Lack of contrast is a common problem encountered when doing TEM of cultured cells, especially of membranes. Using ferrocyanide as a post-fixative can greatly improve membrane fixation and staining. This protocol has been used to study Caco-2 cells grown on PET membranes (the “Caco model”). Caco-2 is a colon cancer cell line that differentiates upon reaching confluency. This allows permeability studies on a cell model, which is reasonably similar to the human intestine.

Basically, the protocol is classical, the only peculiarity consisting in including ferrocyanide in post-fixation. I describe how I prepare and embed the membrane in order to obtain transverse sections of a cell monolayer because I find this is the only way to obtain regular sections with the cells sticking to the membrane (otherwise the ultrathin section splits between the cell and membrane).