Put coverslip with adherent cells in the sample chamber and wash with PBS. This involves gently spraying the PBS on the coverslip with a pippette, and gently rocking the sample chamber back and forth. This should only require about 0.5 mL of PBS.
Aspirate PBS and fill chamber with 1 mL of 4% formaldehyde. Let sit for 15 minutes at room temperature (in cell prep box, covered with a chem wipe).
There are variances in both time and temperature (some incubate rather than using room temperature), so this is something that could be varied if desired.
Aspirate formaldehyde and wash twice with PBS. Aspirate PBS.
For measurements, fill with HBSS at 37C as is typical with live cell measurements.
Berger research group at the University of Rochester