A. Plating:

1. To sterilize glass coverslips, dip in ethanol and flame.
        We use 22x22x1 mm³ coverslips and put them in 6-well plates.
2. Seed 100,000 cells per well overnight and fix the next day.

B. Fixation:

1. Remove the media and rinse once with PBS.
2. Remove the PBS and immediately add -20°C methanol. (Do not allow the cells to dry.)
3. Put the plate in a -20°C freezer for 5 min.
4. Remove the methanol and add PHEM buffer. Fixed cells are kept at 4°C in PHEM.

C. Antibody incubation:

1. Block with appropriate sera (2.5 to 5%) in PHEM buffer for 1 hr with gentle rocking.
2. Add primary antibody to the blocking buffer and incubate for 1 hr with gentle rocking.
3. Remove and wash 4 x 10 min with PHEM buffer.
4. Add secondary antibody in PHEM buffer with sera and incubate for 30 min with gentle rocking.
5. Remove and wash 4 x 10 min with PHEM buffer.

D. Mounting:

1. Pick up coverslip with forceps and drain away excess buffer (can gently aspirate if desired).
2. Put ~20 µl "antifade" on slide and gently lay coverslip on top.
3. After removing excess antifade, either by blotting with Kimwipe or aspirating, seal with Sally
   Hansen clear nail polish. (This brand supposedly works better than others.)
4. KEEP IN THE DARK AT ALL TIMES.
5. Store in -20°C freezer.

   Solutions:

   PHEM buffer:
   25 mM HEPES
   10 mM EGTA
   60 mM PIPES
   2 mM MgCl₂
   pH = 6.9
   (Add in this order.)
   Antifade: 1 ml
   1 mg p-phenylene diamine hydrochloride
   Dissolve in 0.1 ml 10x PBS (20 min at RT)
   Add 0.9 ml 100% glycerol
   Keep covered at all times and no vortexing.
   If it turns brown, it’s no good.
   Aliquot and store at -70°C.