Use 8-chamber poly-d-lysine coated slides.
Plate cells dilutely for differentiation (100 ul from confluent plate to 400 ul complete DMEM media).
Incubate overnight, allowing cells to adhere.
Be gentle for all subsequent steps. Add liquids slowly above one corner of chamber. Do not let cells dry out.
Remove medium and replace with 500 ul serum-free DMEM media for differentiation.
Incubate for desired amount of time (18-36 hr. for differentiation).
Fix cells by incubation for 15 min at room temperature with 100 µl of freshly prepared 4% paraformaldehyde in warm PBS.
Wash cells twice with 500 µl warm PBS.
Permeabilize cells by incubation at RT for 5 min with freshly prepared PBS/1.0% Triton X-100.
Wash once with 500 µl PBS/2% goat serum.
Block with 500 ul PBS/2% goat serum/0.1% Triton X-100.
Incubate for one hour at RT with primary antibody diluted in 100 µl PBS/2% goat serum/0.1% Triton X-100.
Wash twice with 500 µl PBS/2% goat serum.
Incubate for one hour at RT with secondary antibody diluted in 100 µl PBS/2% goat serum/0.1% Triton X-100.
Remove medium.
Incubate for 10 minutes with 1 µg/ml DAPI in 100 µl PBS/2% goat serum.
Wash three times with 500 µl PBS/2% goat serum.
Wash once with 500 µl water.
Remove top of chamber slide. Remove all water and air-dry briefly.
Add one small drop of Vectashield per chamber. (The well-dividers are slightly raised and bubbles form unless there is an excess of Vectasheild.)
Mount coverslip (avoid bubbles) and seal with nail polish.