ICC Protocol

PREPARATION OF EQUIPMENT AND REAGENT

• Equipment

1. A stainless steel pressure cooker or a microwave oven or an electric heater 
2. A water bath pot

• Reagent

1. APES or POLY-L-LYSINE
2. Dimethylbenzene, absolute ethyl and ethanol
3. PBS buffer (pH7.2-7.4)
4. 3% H₂O₂ solution
5. 0.01 mole / L citrate buffer (pH6.0) or 0.5 mole / L EDTA buffer (pH8.0)
6. Enzymatic digestion solution: 0.1% trypsinase or 0.4% pepsase
7. Normal goat serum
8. TBS or PBS (pH9.0-9.5) for fluorescence microscope, (pH7.0-7.4) for optical microscope

ASSAY PROCEDURE

1. For adherent cells, delete the supernatant, add 100 μl of 4% paraformaldehyde into each well, fix for 30 min at RT (In winter, incubate at 37℃ incubator). Note: 4% paraformaldehyde should be freshly prepared with PBS before use.
2. Remove the 4% paraformaldehyde solution, add 200 μl of PBS buffer (pH 7.2~7.4) into each well. Wash with the PBS buffer for 2 times, 5 minutes for each.
3. Add 100 μl of 0.25% Triton X-100 into each well, incubate for 30 min at RT (In winter, use the 37℃ incubator).
4. Wash with PBS buffer (pH 7.2~7.4) for 2 times, 5 minutes for each.
5. Add 60 μl of 10% normal goat serum (blocking solution) into each well, block at 37℃ for 1 hour.
6. Add 50 μl of properly diluted primary antibody into each well, incubate at 4˚C overnight. Wash with PBS (pH 7.2~7.4) 3 times for 5 minutes each. (The primary antibody concentration, incubation time and temperature directly affect the staining efficiency and background intensity. If the positive staining is too weak, the concentration of the primary antibody and the incubation time can be increased; if the background is too high, the primary antibody concentration and the incubation time can be decreased.)
7. Add 50 μl of properly diluted fluorescence conjugated secondary antibody into each well and incubate at 37˚C for 1 hour.
8. Wash with PBS (pH 7.2~7.4) 3 times for 5 minutes each.
9. Observe under fluorescence microscope.