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Posted on Thursday, October 16, 2003

Procedures for cell plating, fixing, antibody incubation and mounting

A. Plating:

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

B. Fixation:

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

C. Antibody incubation:

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

D. Mounting:

Pick up coverslip with forceps and drain away excess buffer (can gently aspirate if desired).
Put ~20 l "antifade" on slide and gently lay coverslip on top.
After removing excess antifade, either by blotting with Kimwipe or aspirating, seal with clear nail polish.

Store in -20C freezer.

PHEM buffer:
10 mM EGTA
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, its no good.
Aliquot and store at -70C.



Submitted by: manny440

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