IImmunofluorescence Protocol

1. Fix cell in 1-4% formaldehyde in growth medium or Phosphate Buffer (PBS w/o salt) or PHEM buffer for 20 min.

1a. Place cells on poly-l-lysine coated slide (1mg/ml poly-lysine) for a period of time. Place a drop of fixative (same as above) on cells.

2. Wash cells in growth medium, Phosphate Buffer or PHEM, 2X with gentle centrifugation or suction.

3. Wash Cells with PBS or with PHEM-DMOS, 2-3X for 5 minutes each wash.

4. Place a drop of cells onto poly-l-lysine slides (if not already on). Let cells sit for 1/2hr, then remove excess fluid and allow to air dry (~5min).

5. Add 1/50 to 1/100 dilution of primary anti-body in PBS or PHEM-DMSO with 0.1% BSA to cells on slide. Incubate at 37 o C for 30minutes.

6. Suction off primary antibody and wash slide in PBS (or PHEM-DMSO) 3X for 5 minutes each.

7. Ass 1/50 to 1/100 dilution of secondary antibody in PBS (or PHEM-DMSO). Incubate at 37 o C for 30 minutes.

8. Suction off secondary antibody and wash slide with PBS (or PHEM-DMSO), 3X for 5 min.

9. Place mountant fluid on each well with cells, place coverslip over cells. Allow for diffusion of mountant for 1 minute and seal with nail polish.

10. Store in refrigerator