1) Start a 2ml culture of cells in the appropriate media. Grow overnight at 30C.

2) Inoculate 5ml of appropriate media with ~300ul of overnight culture. Grow at 30C to log phase (3-4 hours).

3) Fix the cells by adding 600ul of 37% formaldehyde (chemical cabinet under the hood) directly to the culture. Incubate at 30C for 30min.

4) Transfer cells to a 15ml conical tube and pellet by centrifuging 3min at 3000rpm.

5) Aspirate supernatant. Wash cells with 1ml 0.1M potassium phosphate pH 6.5 (premade above centrifuge, filter before use).

6) Repellet cells, aspirate, and wash with 1ml P solution (premade above centrifuge, filter before use).

7) Repellet cells, aspirate, and wash with 1ml P solution (premade above centrifuge, filter before use). Transfer to a microfuge tube.

8) Add 25ul of 1M DTT (door of -20C). Incubate at 30C for 10min.

9) To digest the cell wall, add 15ul of 10mg/ml zymolyase and 5ul of B-mercaptoethanol. Incubate at 30C for 10min.

10) While the cells are digesting, prepare the slide. Put 50ul of 0.1% polylysine (0.3% is aliquoted at -20C, dilute to 0.1% in water, 1:3) on each well. Incubate for several minutes. Wash once with water and allow to air dry. Slides may be prepared several hours in advance.

11) Pellet cells, aspirate supernatant and resuspend in 1ml of P solution. The volume can be varied depending on the desired density of cells on the slide.

12) Place 25ul of the cell suspension onto each well of the prepared slide. Aspirate excess after several minutes.

13) Immediately immerse slide in ice-cold methanol for 6min. Remove and immerse in ice-cold acetone for 30sec. Allow slides to air dry.

14) Beginning with this step, never allow the wells to dry out. Wash the slides once with PBS-BSA (5mg/ml powdered bovine serum albumin in 1X PBS). Aspirate and add primary antibody (diluted in PBS-BSA). Incubate in a moist chamber overnight at 4C.

15) Aspirate and wash the wells four times with PBS-BSA. Add secondary antibody (preferably texas red, diluted in PBS-BSA) to wells. Incubate in moist chamber for at least 2 hours.

16) Aspirate and wash the wells three times with PBS-BSA. Wash one time with PBS. Dilute the DAPI 1:1000 in 1X PBS. Add to wells. Incubate for at least 30sec. Aspirate and wash three times with 1X PBS. Aspirate and allow the slide to air dry.

17) Place slide on a paper towel and wear gloves (p-phenylenediamine is toxic). Place 25-50ul of antifade solution (1-2 flakes p-phenylenediamine in 100ul 10X PBS. Vortex until flakes dissolve. Add 900ul 100% glycerol. Vortex) in alternating wells. Smush with a long coverslip. Wipe up excess antifade solution. Seal edges with several layers of clear nail polish. Keep the slide in a paper towel tent while the polish is drying.

18) Slides can be stored in the dark at -20C for several weeks without loss of signal.