1. Add 100μl of the cells to each well of the Poly L-Lysine (Sigma Cat.#P8920) coated chamber slide (Fisher Cat.#12-565-8). See below for method of preparing slides.

2. Incubate the chamber slide at 37°C/5% CO₂ for 30 minutes. Protect from the light by putting the slide in a small box or covering with foil.
   Note: For all subsequent steps, simply remove by pipet (with a disposable transfer pipet, for example) the first solution and add approximately 500μl of the next solution.

3. Wash three times with PBS.

4. Fix cells by incubating in 4% paraformaldehyde solution for 30 minutes at room temperature.

5. Wash three times with PBS.
   Note: The slide may now be place on a lab bench for all subsequent steps.

6. Permeabilize by incubating with 100% ice cold (-20°C) methanol for 5 minutes.

7. Wash three times with PBS.

8. Block by incubating for 45 minutes with Blocking Solution.

9. Prepare primary antibody: To 1.998 ml Antibody Diluent, add 2.0μl of Anti-Cytochrome c mAb or a control mAb. The stock concentrations of antibody are 1.0mg/ml so the final concentration of this 1:1,000 dilution is 1.0μg/ml. (Optimization is needed for different cell lines.)

10. Incubate in primary antibody or control for one hour in a container covered with foil.

11. Wash three times with PBS, for 5 minutes each, in a container covered with foil.

12. Incubate in donkey anti-mouse FITC conjugate [Jackson ImmunoResearch Cat.#715-095-150] for 60 minutes diluted in PBS. The final concentration of the conjugate antibody should be 1.3μg/ml.

13. Wash three times with PBS, for 10 minutes each, in a container covered with foil.

14. Remove the chamber divider following manufacturer’s directions, mount with Vectashield plus DAPI, and cover with a coverslip. View by fluorescent microscopy.
    

Blocking Solution (5% donkey serum): To 95ml 1X PBS, add 5.0ml donkey serum (Jackson ImmunoResearch Cat.#017-000-121). This solution may be stored at -20°C for up to 3 months. No loss of usefulness has been seen after repeated freeze/thaw cycles.

Antibody Diluent (1% donkey serum): To 10ml Blocking Solution, add 40ml 1X PBS. This solution may be stored at -20°C for up to 3 months. No loss of usefulness has been seen after repeated freeze/thaw cycles.

Prepare poly-L-Lysine (PLL) solution and coat an 8 well chamber slide under sterile conditions in a laminar flow hood.

1. Dilute PLL 1:10 with PBS: To 4.5ml PBS, add 0.5ml PLL.

2. Add 200μl 1:10 PLL solution to each well.

3. Allow to coat for 60 minutes.

4. Wash once with 500μl PBS.

5. Allow to dry for approximately 3 hours. The slides can be stored indefinitely at room temperature under conditions to maintain sterility.