Good thing takes time

Cell lines: 3T3 cell

1. Fix in 4 % para-or Formaldehyde (16 % stock EM grade) in PBS for 20 mintues.
2. Permeabilize in TBS-0.5 % TX for 10 minutes.
3. Block in 10 %FBS/PBS for 10 min-overnight (4oC).
4. Incubate in Fluorescent-phalloidin (1ug/ml from 1mg/ml frozen stock) for 20 minutes.
Do not incubate for longer than 20 minutes; thoses things are usually sticky and will increase background staining.
5. Wash in TBS-0.1 % TX
6. Incubate in 1-10 ug/ml DAPI or Hoesht in 10%FBS/PBS for 10 min
7. Wash in TBS-0.1 %TX
8. Mount, Seal

Solution:

1.
Fix solution:
16 % paraformaldehyde 10ml
10X PBS 4ml
H2O 26ml

2.
Phalloidin solution
Phalloidin (invitrogen, 300U/1.5 methanol) 1:500 dilution
10 % Saponin 1: 100 dilution
10 % FBS/PBS as solvent