A.        Soultions and Reagents

1.         10× PBS: To prepare 1L add 80g NaCl, 2g KCl, 2g KH₂PO₄ and 28.5g NaHPO₄ to 1L water for injection. Adjust pH to 7.4.

2.         4% Polyoxymethylene: To prepare 100mL add 4g polyoxymethylene to 100mL 1×PBS. Adjust pH to 7.4.

3.         1×PBS/0.2% Triton X-100(PBS/Triton):To prepare 500mL add 1mL Triton X-100 to 500mL 1× PBS.

4.         1×PBS/3% BSA(PBS/BSA): To prepare 100mL add 3g BSA to 100mL 1× PBS.

B.        Preparation Fixation permeabilization

1.         Rinse cells briefly in PBS.

2.         Aspirate PBS, cover cells to a depth of 2-3mm about 200ul with 4% polyoxymethylene.

3.         Allow cells to fix for 15 minutes at room temperature.

4.         Aspirate fixative, rinse three times in PBS for 5 minutes each.

5.         Aspirate PBS, cover cells to a depth of 2-3mm about 200ul with PBS/Triton for 5 minutes at room temperature.

6.         Aspirate permeability agent rinse three times in PBS for 5 minutes each.

C.        Immunostaining

1.         Gently add 200ul of primary antibody dilutedin PBS/BSAto the 24 well plates each well.

2.         Incubate 60 minutes at 37℃or overnight at 4℃.

3.         Aspirate diluted primary antibody, then rinse three times in PBS for 5 minutes each.

4.         Incubate in fluorochrome-conjugate secondary antibody diluted in PBS/BSA to the 24 well plates 100ul each well for 30 minutes at room temperature in dark.

5.         Aspirate diluted fluorochrome-conjugate secondary antibody, then rinse three times in PBS for 5 minutes each.

6.         Test under fluorescence microscope.