Electron Microscopy Analysis of Cell Monolayers

Cells were grown on hanging polyethylene terephthalate filters with pore size 0.4 µm (Cat No PIHT12R48, Milipore, Merck, Germany) and seeded in different densities allowing them to become a confluent monolayer at days 1, 2 and 4. When confluent, cells were washed in 5% bovine serum albumin (BSA, Cat No A8022, Sigma Aldrich) and incubated with 0.5 mg/ml cationized ferritin (Cat No F7879, Sigma Aldrich) in 5% BSA for 30 min at room temperature. Cells were initially washed in 5% BSA then in 0.2 M cacodylate buffer [Cat No 11653, electron microscopy sciences (EMS)]. Cells were fixed ON in 2.5% glutaraldehyde (Cat No 16210, EMS) in 0.05 M cacodylate buffer and dehydrated according to standard methods [23 (link)]. Cells were osmificated at 1% for 60 min. (Cat No AGR1017, Agar Scientific) and infiltrated with an epoxy resin (Cat No T031, Embed 812 Medium, TAAB) by using propylene oxide as an intermediate (Cat No 20401, EMS). Cells were initially cut as semi thin sections at 1 µm and stained with toluidine blue. Then cut for TEM at 50 nm thickness and stained with uranyl acetate and lead citrate (EMS) as previously described [23 (link)]. All TEM was performed by using a Philips CM100 equipped with an Olympus Veleta camera connected to a workstation with SIS Analysis software (iTEM).