Indirect Immunofluorescence

1. Grow bacteria to an OD₆₂₀ of 0.8.
2. Wash 3x in PBS containing 2% FCS.
3. Resuspend to the original volume with 1mM MgCl₂. Dispense 0.8 ml into Eppendorf tubes.
4. Add monoclonal or polyclonal serum and vortex (3 on Vortex Genie Fisher)
5. Incubate for 45 min. at 37ºC with agitation.
6. Wash 3x with PBS containing 1% FCS, 1 mM MgCl₂. (Microfuge for 1 min)
7. Incubate at 37ºC for 45 min in PBS containing 2% FCS and a 100-1000 fold dilution of rabbit anti-mouse IgG.
8. Repeat step 6 and then step 7 this time with goat anti-rabbit Ab coupled to FITC.
9. Wash with PBS.
10. Add 10 µl to a slide. Add a cover slip and with a fluorescence microscope measure the fluorescence emitted at 525 nm.

Another method uses bacteria fixed on slides. For each incubation step place the slides in a humid chamber for 10 min. at 37C. The slides are washed by dipping in PBS.