Immunofluorescence Assays
HrpN detection on Ech 3937 cells - modified from Current Protocols in Molecular Biology - France Yap
1. Grow culture on agar or in liquid medium, wash 1 loopful of cells twice with 1 ml of 1 X PBS (pH 7.4).
2. Dilute cells 1:5 in 1 X PBS (200 ul cells plus 800 ul 1 X PBS).
3. Make three to six 1:2 serial dilutions of cells. Pipet 10 ul from each dilution in a drop onto a poly-L-lysine coated glass slide (from Electron Microscopy Sciences #63410).
4. Place the slide into a humid chamber and let the cells settle for 30 min.
5. Immerse the slide into a glass staining dish filled with 4% PFA. Allow cells to fix for 20 min at room temp. (PFA = 1 X PBS saturated paraformaldehyde. Store the paraformaldehyde at 4C and read the MSDS prior to use)
6. Transfer the slide to a new dish containing 3 X PBS and inoculated for 2 min at room temp to stop the cell fixation.
7. Transfer the slide to a new dish containing 1 X PBS and incubate for 2 min. Repeat this step once with fresh 1 X PBS.
8. Dehydrate the slides through a series of washes in 50% ethanol, 70% ethanol, 95% ethanol, and 100% ethanol (5 min for each wash step).
9. Air dry the slides, then store them in a box containing desiccant at -70C until used. The slides must be completely dry before storage.
10. Block the slides with 2% BSA in 1 X PBS for 1 hour.
11. Rinse the slides twice with water and once with 1 X PBS.
12. Incubate with 50 ul of anti-HrpN (1:100 dilution) antibody for 1 hour in a moist chamber.
13. Rinse once with water (in a beaker) , then twice with 1 X PBS. For the PBS wash, use a 15 mm dish on the belly dancer shaker for 5 min)
14. Incubate the slide with 50 ul of FITC labeled secondary antibody diluted 1:100 for 1 hour. (FITC-conjugated anti-rabbit IgG, Molecular Probes, 2 mg/ml #F-2765)
15. Rinse once with water (in a beaker) and twice with 1 X PBS. For the PBS wash, use a 15 mm dish on the belly dancer shaker for 5 min)